Structured Review

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Levels of exosomes in urine of hypertensive patients. (A) There were no differences among the groups in percent of urinary PL-VAP + <t>/CD31</t> + , PL-VAP + /CD144 + , and PL-VAP + /CD31 + /CD144 + exosomes. (B) PTC-EMPs were identified using flow cytometry as PL-VAP + /CD31 − /CD144 − as shown in representative fluorescent images. Scale bar =20 µm. (C) Renal vein and systemic levels of PL-VAP + /CD31 − /CD144 − EMPs were not different among the groups, whereas their urinary levels were elevated in both EH and RVH compared to HVs (p
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Images

1) Product Images from "Loss of Renal Peritubular Capillaries in Hypertensive Patients is Detectable by Urinary Endothelial Microparticle Levels"

Article Title: Loss of Renal Peritubular Capillaries in Hypertensive Patients is Detectable by Urinary Endothelial Microparticle Levels

Journal: Hypertension (Dallas, Tex. : 1979)

doi: 10.1161/HYPERTENSIONAHA.118.11766

Levels of exosomes in urine of hypertensive patients. (A) There were no differences among the groups in percent of urinary PL-VAP + /CD31 + , PL-VAP + /CD144 + , and PL-VAP + /CD31 + /CD144 + exosomes. (B) PTC-EMPs were identified using flow cytometry as PL-VAP + /CD31 − /CD144 − as shown in representative fluorescent images. Scale bar =20 µm. (C) Renal vein and systemic levels of PL-VAP + /CD31 − /CD144 − EMPs were not different among the groups, whereas their urinary levels were elevated in both EH and RVH compared to HVs (p
Figure Legend Snippet: Levels of exosomes in urine of hypertensive patients. (A) There were no differences among the groups in percent of urinary PL-VAP + /CD31 + , PL-VAP + /CD144 + , and PL-VAP + /CD31 + /CD144 + exosomes. (B) PTC-EMPs were identified using flow cytometry as PL-VAP + /CD31 − /CD144 − as shown in representative fluorescent images. Scale bar =20 µm. (C) Renal vein and systemic levels of PL-VAP + /CD31 − /CD144 − EMPs were not different among the groups, whereas their urinary levels were elevated in both EH and RVH compared to HVs (p

Techniques Used: Flow Cytometry, Cytometry

2) Product Images from "A stromal cell niche sustains ILC2-mediated type-2 conditioning in adipose tissue"

Article Title: A stromal cell niche sustains ILC2-mediated type-2 conditioning in adipose tissue

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20190689

Adipose resident IL-33 + cells are MSCs. (A) Gating strategy. CD45 – EpCAM + epithelial cells (Epi), CD45 – CD31 + endothelial cells (End), and CD45 – EpCAM – CD31 – PDGFRα + CD34 + stromal cells. SSC-A, side scatter area; FSC-A, forward scatter area. (B) Proportion of Il33- citrine–positive cells in tissues from Il33 cit/+ mice ( n = 4, representative of two similar independent experiments). (C) Proportion of Il33- citrine + or– CD45 – EpCAM – PDGFRα + stromal cells among live cells in indicated tissues ( n = 4). (D) Histology of WT or Il33 cit/+ mesentery: tomato lectin stain of capillary lumen. Scale bars, 50 µm. (E) Western blot analysis of IL-33 protein from purified WAT-MSCs. Full-length mouse IL-33 (IL-33-FL) in lysate of HEK cells expressing recombinant IL-33 and truncated mouse IL-33 (processed, IL-33-P). Representative of two similar independent experiments. (F) Phenotyping of Il33- citrine + stromal cells. (G) Principal component analysis (PCA) of RNA-seq data from indicated cell populations ( n = 3). (H) Gene expression data (reads per kilobase of transcript per million mapped reads; RPKM). Representative of at least two repeat experiments. (I) Comparison of Il33 + and Il33 – WAT-MSCs from adipose tissue. Genes of interest are highlighted ( n = 3). (J) Adipose differentiation determined by lipid droplet analysis. Representative of two experiments. Scale bars, 100 µm. (K) Myocyte differentiation determined by α-smooth muscle actin (αSMA) staining. Representative of three experiments. Scale bars, 100 µm. Data are represented as mean ± SEM. Max, maximum.
Figure Legend Snippet: Adipose resident IL-33 + cells are MSCs. (A) Gating strategy. CD45 – EpCAM + epithelial cells (Epi), CD45 – CD31 + endothelial cells (End), and CD45 – EpCAM – CD31 – PDGFRα + CD34 + stromal cells. SSC-A, side scatter area; FSC-A, forward scatter area. (B) Proportion of Il33- citrine–positive cells in tissues from Il33 cit/+ mice ( n = 4, representative of two similar independent experiments). (C) Proportion of Il33- citrine + or– CD45 – EpCAM – PDGFRα + stromal cells among live cells in indicated tissues ( n = 4). (D) Histology of WT or Il33 cit/+ mesentery: tomato lectin stain of capillary lumen. Scale bars, 50 µm. (E) Western blot analysis of IL-33 protein from purified WAT-MSCs. Full-length mouse IL-33 (IL-33-FL) in lysate of HEK cells expressing recombinant IL-33 and truncated mouse IL-33 (processed, IL-33-P). Representative of two similar independent experiments. (F) Phenotyping of Il33- citrine + stromal cells. (G) Principal component analysis (PCA) of RNA-seq data from indicated cell populations ( n = 3). (H) Gene expression data (reads per kilobase of transcript per million mapped reads; RPKM). Representative of at least two repeat experiments. (I) Comparison of Il33 + and Il33 – WAT-MSCs from adipose tissue. Genes of interest are highlighted ( n = 3). (J) Adipose differentiation determined by lipid droplet analysis. Representative of two experiments. Scale bars, 100 µm. (K) Myocyte differentiation determined by α-smooth muscle actin (αSMA) staining. Representative of three experiments. Scale bars, 100 µm. Data are represented as mean ± SEM. Max, maximum.

Techniques Used: Mouse Assay, Staining, Western Blot, Purification, Expressing, Recombinant, RNA Sequencing Assay

3) Product Images from "Endothelial mTOR maintains hematopoiesis during aging"

Article Title: Endothelial mTOR maintains hematopoiesis during aging

Journal: bioRxiv

doi: 10.1101/2020.03.13.990911

Aging is associated with decreased mTOR signaling within the bone marrow microenvironment. (A) Representative immunoblot images demonstrating decreased expression of phospho-S6 and phsopho-4EBP-1 in BM cells of aged mice as compared to young mice. (B) Densitometry based quantification of indicated proteins in the BM of aged mice as compared to young mice (n=6 mice per cohort). Expression of Actb was used for normalization. Data represents combined analysis of 2 independent experiments. (C, D) Quantification of mean fluorescent intensity (MFI) of phospho-mTOR (Ser2448), phospho-AKT (Ser473) and phospho S6 (Ser235/236) by Phospho Flow cytometry in (C) Lin-CD45+ HSPCs and (D) Lin-CD45-CD31+VECAD+ BMECs (n=5 mice per cohort). (E) Representative immunoblot images demonstrating decreased expression of phospho-S6 and phsopho-4EBP-1 in BM cells of aged mice treated with Rapamycin. (F) Densitometry based quantification of indicated proteins in the BM of aged mice treated with Rapamycin as compared to aged control mice (n=3 mice per cohort). Expression of Actb was used for normalization. (G-I) Analysis of wild-type cre - (N=3) and CDH5-creERT2 + (N=3) mice revealed no significant differences in (G) BM cellularity, (H) HSC frequency, and (I) peripheral blood lineage composition indicating that endothelial-specific expression of cre-ERT2 transgene does not affect hematopoiesis. (J-L) Hematopoietic analysis of heterozygote mTOR fl/+ cre-ERT2+ (N=5) and mTOR (ECKO) (N=5) demonstrated that unlike mTOR (ECKO) mice, the littermate heterozygote mTOR fl/+ cre-ERT2+ mice do not manifest increased BM cellularity (J) , increased HSC frequency (K) , and myeloid-skewed peripheral blood lineage composition (L) . All mice utilized in (G-L) were administered 200 mg/kg tamoxifen via intraperitoneal injection at a concentration of 30 mg/mL in sunflower oil on three consecutive days, followed by three days off, and three additional days of injection. Note that the same regimen induces HSPC aging phenotypes in homozygote mTOR fl/fl cre-ERT2+ ( Figures 3 , 4 ), indicating that loss of both alleles of endothelial mTOR are essential to induce HSPC aging phenotypes. Error bars represent mean ± SEM. Statistical significance determined using Student’s t-test. *P
Figure Legend Snippet: Aging is associated with decreased mTOR signaling within the bone marrow microenvironment. (A) Representative immunoblot images demonstrating decreased expression of phospho-S6 and phsopho-4EBP-1 in BM cells of aged mice as compared to young mice. (B) Densitometry based quantification of indicated proteins in the BM of aged mice as compared to young mice (n=6 mice per cohort). Expression of Actb was used for normalization. Data represents combined analysis of 2 independent experiments. (C, D) Quantification of mean fluorescent intensity (MFI) of phospho-mTOR (Ser2448), phospho-AKT (Ser473) and phospho S6 (Ser235/236) by Phospho Flow cytometry in (C) Lin-CD45+ HSPCs and (D) Lin-CD45-CD31+VECAD+ BMECs (n=5 mice per cohort). (E) Representative immunoblot images demonstrating decreased expression of phospho-S6 and phsopho-4EBP-1 in BM cells of aged mice treated with Rapamycin. (F) Densitometry based quantification of indicated proteins in the BM of aged mice treated with Rapamycin as compared to aged control mice (n=3 mice per cohort). Expression of Actb was used for normalization. (G-I) Analysis of wild-type cre - (N=3) and CDH5-creERT2 + (N=3) mice revealed no significant differences in (G) BM cellularity, (H) HSC frequency, and (I) peripheral blood lineage composition indicating that endothelial-specific expression of cre-ERT2 transgene does not affect hematopoiesis. (J-L) Hematopoietic analysis of heterozygote mTOR fl/+ cre-ERT2+ (N=5) and mTOR (ECKO) (N=5) demonstrated that unlike mTOR (ECKO) mice, the littermate heterozygote mTOR fl/+ cre-ERT2+ mice do not manifest increased BM cellularity (J) , increased HSC frequency (K) , and myeloid-skewed peripheral blood lineage composition (L) . All mice utilized in (G-L) were administered 200 mg/kg tamoxifen via intraperitoneal injection at a concentration of 30 mg/mL in sunflower oil on three consecutive days, followed by three days off, and three additional days of injection. Note that the same regimen induces HSPC aging phenotypes in homozygote mTOR fl/fl cre-ERT2+ ( Figures 3 , 4 ), indicating that loss of both alleles of endothelial mTOR are essential to induce HSPC aging phenotypes. Error bars represent mean ± SEM. Statistical significance determined using Student’s t-test. *P

Techniques Used: Expressing, Mouse Assay, Flow Cytometry, Injection, Concentration Assay

4) Product Images from "Endothelial-specific inhibition of NF-κB enhances functional haematopoiesis"

Article Title: Endothelial-specific inhibition of NF-κB enhances functional haematopoiesis

Journal: Nature Communications

doi: 10.1038/ncomms13829

Tie2::IκB-SS mice do not alter NF-κB signalling in HSCs. Phenotypic haematopoietic stem cells (HSCs) and bone marrow endothelial cells (BMECs) from adult Tie2::IκB-SS and littermate control mice (12–16 weeks) were assessed for off-target expression. ( a ) Phenotypic HSCs (cKIT + Lineage − SCA1 + CD150 + CD48 − ) and ( b ) BMECs (VECAD + CD31 + CD45 − ) from control and Tie2::IκB-SS mice were assessed for TIE2 expression and NF-κB signalling capacity (p65; phosphorylated Ser536) by flow cytometry. To assess HSC functionality in Tie2::IκB-SS derived WBM, reciprocal transplantations ( Tie2::IκB-SS WBM into wild-type (WT) recipients or WT WBM into Tie2::IκB-SS recipients) were established and assayed four months post-transplantation. ( c , d ) Quantification of ( c ) total haematopoietic cell counts per femur ( n =3) and ( d ) frequency of phenotypic HSCs following reciprocal transplantations ( n =3). ( e ) Time course of haematopoietic recovery from inverse transplantations following myelosuppressive irradiation ( n =7). ( f ) Representative images of intravitally labelled vasculature (VECAD) from reciprocal transplantations following myelosuppression. Scale bar, 100 μm. Error bars represent mean±s.e.m. * P
Figure Legend Snippet: Tie2::IκB-SS mice do not alter NF-κB signalling in HSCs. Phenotypic haematopoietic stem cells (HSCs) and bone marrow endothelial cells (BMECs) from adult Tie2::IκB-SS and littermate control mice (12–16 weeks) were assessed for off-target expression. ( a ) Phenotypic HSCs (cKIT + Lineage − SCA1 + CD150 + CD48 − ) and ( b ) BMECs (VECAD + CD31 + CD45 − ) from control and Tie2::IκB-SS mice were assessed for TIE2 expression and NF-κB signalling capacity (p65; phosphorylated Ser536) by flow cytometry. To assess HSC functionality in Tie2::IκB-SS derived WBM, reciprocal transplantations ( Tie2::IκB-SS WBM into wild-type (WT) recipients or WT WBM into Tie2::IκB-SS recipients) were established and assayed four months post-transplantation. ( c , d ) Quantification of ( c ) total haematopoietic cell counts per femur ( n =3) and ( d ) frequency of phenotypic HSCs following reciprocal transplantations ( n =3). ( e ) Time course of haematopoietic recovery from inverse transplantations following myelosuppressive irradiation ( n =7). ( f ) Representative images of intravitally labelled vasculature (VECAD) from reciprocal transplantations following myelosuppression. Scale bar, 100 μm. Error bars represent mean±s.e.m. * P

Techniques Used: Mouse Assay, Expressing, Flow Cytometry, Cytometry, Derivative Assay, Transplantation Assay, Irradiation

Endothelial-specific NF-κB suppression enhances haematopoietic recovery. Adult Tie2::IκB-SS and littermate control mice (12–16 weeks) were subjected to 650 Rads of myelosuppressive irradiation and assayed for haematopoietic recovery. ( a ) NF-κB signalling in in vivo BMECs (VECAD + CD31 + CD45 − ) was quantified by mean fluorescence intensity (MFI) of phosphorylated p65 (Ser536) ( n =3). ( b ) Time-course of haematopoietic recovery in peripheral blood ( n =14, control; n =19, Tie2::IκB-SS ). ( c , d ) Quantification of total haematopoietic cells ( c ) and phenotypic haematopoietic stem and progenitor cells ( d ) (HSPCs; cKIT + Lineage − SCA1 + ) ( n =7, control; n =6, Tie2::IκB-SS ). ( e ) Representative images of femurs from Col2.3::GFP ; Tie2::IκB-SS and Col2.3::GFP control mice at steady state and following sublethal irradiation. Intravitally labelled vasculature (VECAD; red), Col1a1 expressing osteoblasts (green) and nuclear staining (DAPI; blue) is noted. Scale bar, 100 μm and 200 μm, respectively. ( f ) Representative images of intravitally labelled vasculature (VECAD; red), megakaryocytes (green; citrulline) and nuclear staining (DAPI; blue). Dashed line demarcates bone. Scale bar, 100 μm. ( g , h ) Representative images of femurs stained with antibodies raised against PLIN1 ( g ; green) or LEPR ( h ; green), intravitally labelled vasculature (VECAD; red) and nuclear staining (DAPI; blue). Dashed line demarcates bone. Scale bar, 100 μm and 200 μm, respectively. ( i ) Representative images and ( j ) quantification of VEGFR3 + (brown) sinusoidal BM endothelial damage following irradiation (650 Rads). Type I haemorrhagic (asterisk), Type I discontinuous (red arrow) and Type II regressed (yellow arrow) femoral vessels are noted ( n =3 per group). Sections were counterstained with Haematoxylin (blue). Scale bar, 200 μm. Error bars represent mean±s.e.m. ( a ) * P
Figure Legend Snippet: Endothelial-specific NF-κB suppression enhances haematopoietic recovery. Adult Tie2::IκB-SS and littermate control mice (12–16 weeks) were subjected to 650 Rads of myelosuppressive irradiation and assayed for haematopoietic recovery. ( a ) NF-κB signalling in in vivo BMECs (VECAD + CD31 + CD45 − ) was quantified by mean fluorescence intensity (MFI) of phosphorylated p65 (Ser536) ( n =3). ( b ) Time-course of haematopoietic recovery in peripheral blood ( n =14, control; n =19, Tie2::IκB-SS ). ( c , d ) Quantification of total haematopoietic cells ( c ) and phenotypic haematopoietic stem and progenitor cells ( d ) (HSPCs; cKIT + Lineage − SCA1 + ) ( n =7, control; n =6, Tie2::IκB-SS ). ( e ) Representative images of femurs from Col2.3::GFP ; Tie2::IκB-SS and Col2.3::GFP control mice at steady state and following sublethal irradiation. Intravitally labelled vasculature (VECAD; red), Col1a1 expressing osteoblasts (green) and nuclear staining (DAPI; blue) is noted. Scale bar, 100 μm and 200 μm, respectively. ( f ) Representative images of intravitally labelled vasculature (VECAD; red), megakaryocytes (green; citrulline) and nuclear staining (DAPI; blue). Dashed line demarcates bone. Scale bar, 100 μm. ( g , h ) Representative images of femurs stained with antibodies raised against PLIN1 ( g ; green) or LEPR ( h ; green), intravitally labelled vasculature (VECAD; red) and nuclear staining (DAPI; blue). Dashed line demarcates bone. Scale bar, 100 μm and 200 μm, respectively. ( i ) Representative images and ( j ) quantification of VEGFR3 + (brown) sinusoidal BM endothelial damage following irradiation (650 Rads). Type I haemorrhagic (asterisk), Type I discontinuous (red arrow) and Type II regressed (yellow arrow) femoral vessels are noted ( n =3 per group). Sections were counterstained with Haematoxylin (blue). Scale bar, 200 μm. Error bars represent mean±s.e.m. ( a ) * P

Techniques Used: Mouse Assay, Irradiation, In Vivo, Fluorescence, Expressing, Staining

5) Product Images from "Neonatal thymectomy reveals differentiation and plasticity within human naive T cells"

Article Title: Neonatal thymectomy reveals differentiation and plasticity within human naive T cells

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI84997

Thymic tissue regeneration results in restoration of the naive and effector T cell compartment later in life. ( A ) Lymphocyte count of CD3 + , CD4 + , and CD8 + T cells in HCs and neonatally Tx children older than 10 years of age. ( B ) Percentages of CD31 expression on CD45RA + CD4 + T cells. ( C ) Percentages of naive T cells (CD45RA + CCR7 + ) within the CD4 + T cell population. ( D ) Percentages of Tcm (CD45RA – CCR7 + ). ( E ) Percentages of Tem (CD45RA – CCR7 – ). ( F ) Percentages of Temra (CD45RA + CCR7 – ). ( G ) Percentages of Tscm (CD45RA + CCR7 + CD28 + CD27 + FAS + ) in CD4 + T cells. Black squares (or black bars), older HCs ( n = 10); gray squares (or gray bars), older Tx patients ( n = 24–26). Older Tx patients are further divided in high percentages of CD31 (n = 17–19; closed gray square in B ) and older TX with low CD31 percentages ( n = 7; open gray squares in B ), as described in the x . * P
Figure Legend Snippet: Thymic tissue regeneration results in restoration of the naive and effector T cell compartment later in life. ( A ) Lymphocyte count of CD3 + , CD4 + , and CD8 + T cells in HCs and neonatally Tx children older than 10 years of age. ( B ) Percentages of CD31 expression on CD45RA + CD4 + T cells. ( C ) Percentages of naive T cells (CD45RA + CCR7 + ) within the CD4 + T cell population. ( D ) Percentages of Tcm (CD45RA – CCR7 + ). ( E ) Percentages of Tem (CD45RA – CCR7 – ). ( F ) Percentages of Temra (CD45RA + CCR7 – ). ( G ) Percentages of Tscm (CD45RA + CCR7 + CD28 + CD27 + FAS + ) in CD4 + T cells. Black squares (or black bars), older HCs ( n = 10); gray squares (or gray bars), older Tx patients ( n = 24–26). Older Tx patients are further divided in high percentages of CD31 (n = 17–19; closed gray square in B ) and older TX with low CD31 percentages ( n = 7; open gray squares in B ), as described in the x . * P

Techniques Used: Expressing, Transmission Electron Microscopy

Naive CD4 + T cell functionality is impaired after neonatal thymectomy in early life, but restores after thymic regeneration in later life. ( A ) Left panel, calcium flux of CD4 + naive T cells in young children (young HCs, n = 4 black, young Tx, n = 6 gray, mean ± SD). Right panel, AUC of the calcium flux. ( B ) Representative dot plot of IL-8 production in CD45RA + CD4 + T cell population of young HCs. ( C ) IL-2 production in naive CD4 + T cells of young HCs ( n = 15) and young Tx patients ( n = 10) after PMA/ion stimulation. ( D ) IL-8 production by naive CD4 + T cells of young HC ( n = 19) and young Tx patient ( n = 15) PMA/ion stimulation. ( E ) Left panel, calcium flux of CD4 naive T cells of older HCs and Tx children. Right panel, AUC of the calcium flux (HC 1–5 yr, n = 5; high CD31, Tx > 10 yr, n = 6; low CD31, Tx > 10 yr, n = 1). ( F ) IL-8 production in naive CD4 + T cells of older HC ( n = 10) and Tx patient (high CD31, n = 17; low CD31, n . * P
Figure Legend Snippet: Naive CD4 + T cell functionality is impaired after neonatal thymectomy in early life, but restores after thymic regeneration in later life. ( A ) Left panel, calcium flux of CD4 + naive T cells in young children (young HCs, n = 4 black, young Tx, n = 6 gray, mean ± SD). Right panel, AUC of the calcium flux. ( B ) Representative dot plot of IL-8 production in CD45RA + CD4 + T cell population of young HCs. ( C ) IL-2 production in naive CD4 + T cells of young HCs ( n = 15) and young Tx patients ( n = 10) after PMA/ion stimulation. ( D ) IL-8 production by naive CD4 + T cells of young HC ( n = 19) and young Tx patient ( n = 15) PMA/ion stimulation. ( E ) Left panel, calcium flux of CD4 naive T cells of older HCs and Tx children. Right panel, AUC of the calcium flux (HC 1–5 yr, n = 5; high CD31, Tx > 10 yr, n = 6; low CD31, Tx > 10 yr, n = 1). ( F ) IL-8 production in naive CD4 + T cells of older HC ( n = 10) and Tx patient (high CD31, n = 17; low CD31, n . * P

Techniques Used:

IL-8 production is enriched in the PTK7 + fraction of CD31 + naive CD4 + T cells and lost after cell division. ( A ) Proportion of CD31 + and CD31 – naive CD4 + T cells in the CD4 + T cell compartment of HCs, 1–5 yr ( n = 19) and Tx patients, 1–5 yr ( n = 15). ( B ) Expression of IL-8 by CD31 + and CD31 – naive CD4 + T cells of young HCs ( n = 19) and Tx patients ( n = 15). ( C ) Expression of IL-8 by CD31 + and CD31 – naive CD4 + T cells of older HCs ( n = 10) and older Tx patients separated on the basis of low ( n = 7) or high percentage of CD31 + ( n = 19). ( D ) Paired IL-8 expression measurements by SP CD3 hi CD4 + CD8 – thymocytes and blood CD31 + naive CD4 + T cells (PBMCs) from the Tx neonates ( n = 3). ( E ) IL-8 expression by PTK7 + (black dots) and PTK7 – (gray dots) CD31 + naive CD4 + T cells from young HCs ( n = 5). ( F ) PTK7 expression after each cell division following cytokine stimulation of FACS-sorted CD31 + naive CD4 + T cells from older HCs ( n = 6). ( G ) IL-8 expression after each cell division following cytokine stimulation of FACS-sorted CD31 + naive CD4 + T cells from older HCs ( n . * P
Figure Legend Snippet: IL-8 production is enriched in the PTK7 + fraction of CD31 + naive CD4 + T cells and lost after cell division. ( A ) Proportion of CD31 + and CD31 – naive CD4 + T cells in the CD4 + T cell compartment of HCs, 1–5 yr ( n = 19) and Tx patients, 1–5 yr ( n = 15). ( B ) Expression of IL-8 by CD31 + and CD31 – naive CD4 + T cells of young HCs ( n = 19) and Tx patients ( n = 15). ( C ) Expression of IL-8 by CD31 + and CD31 – naive CD4 + T cells of older HCs ( n = 10) and older Tx patients separated on the basis of low ( n = 7) or high percentage of CD31 + ( n = 19). ( D ) Paired IL-8 expression measurements by SP CD3 hi CD4 + CD8 – thymocytes and blood CD31 + naive CD4 + T cells (PBMCs) from the Tx neonates ( n = 3). ( E ) IL-8 expression by PTK7 + (black dots) and PTK7 – (gray dots) CD31 + naive CD4 + T cells from young HCs ( n = 5). ( F ) PTK7 expression after each cell division following cytokine stimulation of FACS-sorted CD31 + naive CD4 + T cells from older HCs ( n = 6). ( G ) IL-8 expression after each cell division following cytokine stimulation of FACS-sorted CD31 + naive CD4 + T cells from older HCs ( n . * P

Techniques Used: Expressing, FACS

Neonatal thymectomy results in lower naive CD4 + T cell percentages and skewing toward a memory phenotype in the first years (1–5 years) of life. ( A ) Blood lymphocyte count of CD3 + , CD4 + , and CD8 + T cells in HCs and neonatally Tx children for the age groups of 1 to 5 years. ( B ) Left panel, percentages of naive T cells (CD45RA + CCR7 + ) within the CD4 + T cell population. Right panel, percentages of CD31-expressing cells among CD45RA + CD4 + T cells. ( C ) Percentages of Tcm (CD45RA – CCR7 + ), Tem (CD45RA – CCR7 – ), and Temra (CD45RA + CCR7 – ) in the CD4 + T cell pool. ( D ) Percentages of Tscm (CD45RA + CCR7 + CD28 + CD27 + FAS + ) in the CD4 + T cell pool. ( E ) Percentages of naive T cells (CD45RA + CCR7 + ) within the CD8 + T cell population. ( F ) Percentages of Tcm (CD45RA – CCR7 + ), Tem (CD45RA – CCR7 – ), and Temra (CD45RA + CCR7 – ) in the CD8 + T cell pool. Black circles (or black bar, A ), young HCs ( n = 8–14); gray circles (gray bar, A ), young Tx ( n . * P
Figure Legend Snippet: Neonatal thymectomy results in lower naive CD4 + T cell percentages and skewing toward a memory phenotype in the first years (1–5 years) of life. ( A ) Blood lymphocyte count of CD3 + , CD4 + , and CD8 + T cells in HCs and neonatally Tx children for the age groups of 1 to 5 years. ( B ) Left panel, percentages of naive T cells (CD45RA + CCR7 + ) within the CD4 + T cell population. Right panel, percentages of CD31-expressing cells among CD45RA + CD4 + T cells. ( C ) Percentages of Tcm (CD45RA – CCR7 + ), Tem (CD45RA – CCR7 – ), and Temra (CD45RA + CCR7 – ) in the CD4 + T cell pool. ( D ) Percentages of Tscm (CD45RA + CCR7 + CD28 + CD27 + FAS + ) in the CD4 + T cell pool. ( E ) Percentages of naive T cells (CD45RA + CCR7 + ) within the CD8 + T cell population. ( F ) Percentages of Tcm (CD45RA – CCR7 + ), Tem (CD45RA – CCR7 – ), and Temra (CD45RA + CCR7 – ) in the CD8 + T cell pool. Black circles (or black bar, A ), young HCs ( n = 8–14); gray circles (gray bar, A ), young Tx ( n . * P

Techniques Used: Expressing, Transmission Electron Microscopy

6) Product Images from "Induction of Tertiary Lymphoid Structures With Antitumor Function by a Lymph Node-Derived Stromal Cell Line"

Article Title: Induction of Tertiary Lymphoid Structures With Antitumor Function by a Lymph Node-Derived Stromal Cell Line

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01609

Establishing a lymph node (LN)-derived stromal cell line. (A) A photomicrograph of a LN-derived monoclonal stromal cell line (#2) in culture. Monoclonal cell lines were generated by limiting dilution. Scale bar denotes 0.2 mm. (B) Total RNA was extracted from the stromal cell line (#2) at 3 different passages and mRNA level of indicated 11 chemokines were analyzed by mouse genome arrays. Log2 transformed data were presented and red bars denote the mean. (C) The stromal cell line was stained for CD3, CD45, CD31, podoplanin, LTβ receptor (LTβR), and vascular cell adhesion molecule 1 (VCAM-1), and analyzed by flow cytometry. The majority of the cells are fibroblastic reticular cells with expression of VCAM-1 and LTβR.
Figure Legend Snippet: Establishing a lymph node (LN)-derived stromal cell line. (A) A photomicrograph of a LN-derived monoclonal stromal cell line (#2) in culture. Monoclonal cell lines were generated by limiting dilution. Scale bar denotes 0.2 mm. (B) Total RNA was extracted from the stromal cell line (#2) at 3 different passages and mRNA level of indicated 11 chemokines were analyzed by mouse genome arrays. Log2 transformed data were presented and red bars denote the mean. (C) The stromal cell line was stained for CD3, CD45, CD31, podoplanin, LTβ receptor (LTβR), and vascular cell adhesion molecule 1 (VCAM-1), and analyzed by flow cytometry. The majority of the cells are fibroblastic reticular cells with expression of VCAM-1 and LTβR.

Techniques Used: Derivative Assay, Generated, Transformation Assay, Staining, Flow Cytometry, Cytometry, Expressing

7) Product Images from "Neuropilin 1 regulates bone marrow vascular regeneration and hematopoietic reconstitution"

Article Title: Neuropilin 1 regulates bone marrow vascular regeneration and hematopoietic reconstitution

Journal: Nature Communications

doi: 10.1038/s41467-021-27263-y

NRP1 inhibition accelerates BM vascular regeneration in vivo. Representative confocal images of VE-cadherin + (magenta) and CD31 + (green) BM vessels, and nuclei (blue) in femurs from a non-IRR mice and at b day +3 and day +7 following 500 cGy and treatment with IgG (10 µg/dose) or anti-NRP1 (10 µg/dose). Left image shows full view taken using 20× lens and right images display individual channels from magnified yellow box. Scale bar 50 µm; magnified view scale bar, 20 µm. c Quantification of VE-Cadherin vascular area from the images in a , b . Dotted line represents mean Non-IRR vascular area. Data presented as mean values +/− SEM. ( n = 2 independent experiments, d3 IgG n = 4 fields of view, d3 anti-NRP1 n = 5 fields, d7 IgG n = 7 fields, d7 anti-NRP1 n = 7 fields; IgG d3: p = 0.016, anti-NRP1 d3: p = 0.0467, IgG d7: p = 0.0349, anti-NRP1 d7: p = 0.9694. d BM cell counts at day +7 following 500 cGy and treatment with IgG or anti-NRP1 ( n = 5 mice/group, p = 0.0025). Dotted line shows cell counts of non-IRR BM. e At left, representative flow cytometric analysis of CD45 − VE-cad + BM ECs within BM lin − cells from the groups shown. At right, percentages of BM ECs at day +7 following 500 cGy. Dotted line represents %VE-cad + ECs in non-IRR controls ( n = 5 mice/group). f At left, representative histograms of % activated caspase 3/7 + cells within lin − CD45 − VE-cad + BM ECs at day +7 following 500 cGy. At right, mean % activated caspase 3/7 + BM ECs at day +7 following 500 cGy ( n = 5/group). Dotted line represents % activated caspase 3/7 + ECs in non-IRR controls. g Levels of Evans Blue Dye (EBD) in the BM extracellular space at +24 h following 500 cGy and treatment with IgG or anti-NRP1 ( n = 5–6 mice/group, non-IRR vs. IgG: p = 0.0024, IgG vs. anti-NRP1: p = 0.188, non-IRR vs. anti-NRP1 p = 0.2679). d – f Data assessed by Student’s two-tailed t -test, g One-way ANOVA with Holm-Sidak’s multiple comparison two-sided t -test, c Two-sided one-sample t -test and Wilcoxon test used to compare sample means to the mean of non-IRR samples, * p
Figure Legend Snippet: NRP1 inhibition accelerates BM vascular regeneration in vivo. Representative confocal images of VE-cadherin + (magenta) and CD31 + (green) BM vessels, and nuclei (blue) in femurs from a non-IRR mice and at b day +3 and day +7 following 500 cGy and treatment with IgG (10 µg/dose) or anti-NRP1 (10 µg/dose). Left image shows full view taken using 20× lens and right images display individual channels from magnified yellow box. Scale bar 50 µm; magnified view scale bar, 20 µm. c Quantification of VE-Cadherin vascular area from the images in a , b . Dotted line represents mean Non-IRR vascular area. Data presented as mean values +/− SEM. ( n = 2 independent experiments, d3 IgG n = 4 fields of view, d3 anti-NRP1 n = 5 fields, d7 IgG n = 7 fields, d7 anti-NRP1 n = 7 fields; IgG d3: p = 0.016, anti-NRP1 d3: p = 0.0467, IgG d7: p = 0.0349, anti-NRP1 d7: p = 0.9694. d BM cell counts at day +7 following 500 cGy and treatment with IgG or anti-NRP1 ( n = 5 mice/group, p = 0.0025). Dotted line shows cell counts of non-IRR BM. e At left, representative flow cytometric analysis of CD45 − VE-cad + BM ECs within BM lin − cells from the groups shown. At right, percentages of BM ECs at day +7 following 500 cGy. Dotted line represents %VE-cad + ECs in non-IRR controls ( n = 5 mice/group). f At left, representative histograms of % activated caspase 3/7 + cells within lin − CD45 − VE-cad + BM ECs at day +7 following 500 cGy. At right, mean % activated caspase 3/7 + BM ECs at day +7 following 500 cGy ( n = 5/group). Dotted line represents % activated caspase 3/7 + ECs in non-IRR controls. g Levels of Evans Blue Dye (EBD) in the BM extracellular space at +24 h following 500 cGy and treatment with IgG or anti-NRP1 ( n = 5–6 mice/group, non-IRR vs. IgG: p = 0.0024, IgG vs. anti-NRP1: p = 0.188, non-IRR vs. anti-NRP1 p = 0.2679). d – f Data assessed by Student’s two-tailed t -test, g One-way ANOVA with Holm-Sidak’s multiple comparison two-sided t -test, c Two-sided one-sample t -test and Wilcoxon test used to compare sample means to the mean of non-IRR samples, * p

Techniques Used: Inhibition, In Vivo, Mouse Assay, Two Tailed Test

8) Product Images from "Endothelial mTOR maintains hematopoiesis during aging"

Article Title: Endothelial mTOR maintains hematopoiesis during aging

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20191212

Aging is associated with decreased mTOR signaling within the BM microenvironment. (A) Representative immunoblot images demonstrating decreased expression of phospho-S6 and phospho-4EBP-1 in BM cells of aged mice compared with those of young mice. (B) Densitometry-based quantification of indicated proteins in the BM of aged mice compared with young mice ( n = 6 mice per cohort). Expression of Actb was used for normalization. Data represent combined analysis of two independent experiments. (C and D) Quantification of mean fluorescent intensity (MFI) of phospho-mTOR (Ser2448), phospho-AKT (Ser473), and phospho-S6 (Ser235/236) by Phosphoflow cytometry in Lin − CD45 + HSPCs (C) and Lin − CD45 − CD31 + VECAD + BMECs (D; n = 5 mice per cohort). (E) Representative immunoblot images demonstrating decreased expression of phospho-S6 and phospho-4EBP-1 in BM cells of aged mice treated with Rapamycin. (F) Densitometry-based quantification of indicated proteins in the BM of aged mice treated with Rapamycin compared with aged control mice ( n = 3 mice per cohort). Expression of Actb was used for normalization. (G–I) Analysis of wild-type cre - ( n = 3) and C dh 5-creERT2 + ( n = 3) mice revealed no significant differences in BM cellularity (G), HSC frequency (H), and peripheral blood lineage composition (I) indicating that endothelial-specific expression of the creERT2 transgene does not affect hematopoiesis. (J–L) Hematopoietic analysis of heterozygote mTOR fl/+ creERT2 + ( n = 5) and mTOR (ECKO) ( n = 5) demonstrated that unlike mTOR (ECKO) mice, the littermate heterozygote mTOR fl/+ creERT2 + mice do not manifest increased BM cellularity (J), increased HSC frequency (K), and myeloid-skewed peripheral blood lineage composition (L). All mice used in G–L were administered 200 mg/kg tamoxifen via intraperitoneal injection at a concentration of 30 mg/ml in sunflower oil on consecutive 3 d, followed by 3 d off, and an additional 3 d of injection. Note that the same regimen induces HSPC aging phenotypes in homozygote mTOR fl/fl cre-ERT2 + ( Fig. 3 and Fig. 4 ), indicating that loss of both alleles of endothelial mTOR is essential to induce HSPC aging phenotypes. Error bars represent mean ± SEM. Statistical significance determined using Student’s t test. *, P
Figure Legend Snippet: Aging is associated with decreased mTOR signaling within the BM microenvironment. (A) Representative immunoblot images demonstrating decreased expression of phospho-S6 and phospho-4EBP-1 in BM cells of aged mice compared with those of young mice. (B) Densitometry-based quantification of indicated proteins in the BM of aged mice compared with young mice ( n = 6 mice per cohort). Expression of Actb was used for normalization. Data represent combined analysis of two independent experiments. (C and D) Quantification of mean fluorescent intensity (MFI) of phospho-mTOR (Ser2448), phospho-AKT (Ser473), and phospho-S6 (Ser235/236) by Phosphoflow cytometry in Lin − CD45 + HSPCs (C) and Lin − CD45 − CD31 + VECAD + BMECs (D; n = 5 mice per cohort). (E) Representative immunoblot images demonstrating decreased expression of phospho-S6 and phospho-4EBP-1 in BM cells of aged mice treated with Rapamycin. (F) Densitometry-based quantification of indicated proteins in the BM of aged mice treated with Rapamycin compared with aged control mice ( n = 3 mice per cohort). Expression of Actb was used for normalization. (G–I) Analysis of wild-type cre - ( n = 3) and C dh 5-creERT2 + ( n = 3) mice revealed no significant differences in BM cellularity (G), HSC frequency (H), and peripheral blood lineage composition (I) indicating that endothelial-specific expression of the creERT2 transgene does not affect hematopoiesis. (J–L) Hematopoietic analysis of heterozygote mTOR fl/+ creERT2 + ( n = 5) and mTOR (ECKO) ( n = 5) demonstrated that unlike mTOR (ECKO) mice, the littermate heterozygote mTOR fl/+ creERT2 + mice do not manifest increased BM cellularity (J), increased HSC frequency (K), and myeloid-skewed peripheral blood lineage composition (L). All mice used in G–L were administered 200 mg/kg tamoxifen via intraperitoneal injection at a concentration of 30 mg/ml in sunflower oil on consecutive 3 d, followed by 3 d off, and an additional 3 d of injection. Note that the same regimen induces HSPC aging phenotypes in homozygote mTOR fl/fl cre-ERT2 + ( Fig. 3 and Fig. 4 ), indicating that loss of both alleles of endothelial mTOR is essential to induce HSPC aging phenotypes. Error bars represent mean ± SEM. Statistical significance determined using Student’s t test. *, P

Techniques Used: Expressing, Mouse Assay, Cytometry, Injection, Concentration Assay

9) Product Images from "Endothelial mTOR maintains hematopoiesis during aging"

Article Title: Endothelial mTOR maintains hematopoiesis during aging

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20191212

Aging is associated with decreased mTOR signaling within the BM microenvironment. (A) Representative immunoblot images demonstrating decreased expression of phospho-S6 and phospho-4EBP-1 in BM cells of aged mice compared with those of young mice. (B) Densitometry-based quantification of indicated proteins in the BM of aged mice compared with young mice ( n = 6 mice per cohort). Expression of Actb was used for normalization. Data represent combined analysis of two independent experiments. (C and D) Quantification of mean fluorescent intensity (MFI) of phospho-mTOR (Ser2448), phospho-AKT (Ser473), and phospho-S6 (Ser235/236) by Phosphoflow cytometry in Lin − CD45 + HSPCs (C) and Lin − CD45 − CD31 + VECAD + BMECs (D; n = 5 mice per cohort). (E) Representative immunoblot images demonstrating decreased expression of phospho-S6 and phospho-4EBP-1 in BM cells of aged mice treated with Rapamycin. (F) Densitometry-based quantification of indicated proteins in the BM of aged mice treated with Rapamycin compared with aged control mice ( n = 3 mice per cohort). Expression of Actb was used for normalization. (G–I) Analysis of wild-type cre - ( n = 3) and C dh 5-creERT2 + ( n = 3) mice revealed no significant differences in BM cellularity (G), HSC frequency (H), and peripheral blood lineage composition (I) indicating that endothelial-specific expression of the creERT2 transgene does not affect hematopoiesis. (J–L) Hematopoietic analysis of heterozygote mTOR fl/+ creERT2 + ( n = 5) and mTOR (ECKO) ( n = 5) demonstrated that unlike mTOR (ECKO) mice, the littermate heterozygote mTOR fl/+ creERT2 + mice do not manifest increased BM cellularity (J), increased HSC frequency (K), and myeloid-skewed peripheral blood lineage composition (L). All mice used in G–L were administered 200 mg/kg tamoxifen via intraperitoneal injection at a concentration of 30 mg/ml in sunflower oil on consecutive 3 d, followed by 3 d off, and an additional 3 d of injection. Note that the same regimen induces HSPC aging phenotypes in homozygote mTOR fl/fl cre-ERT2 + ( Fig. 3 and Fig. 4 ), indicating that loss of both alleles of endothelial mTOR is essential to induce HSPC aging phenotypes. Error bars represent mean ± SEM. Statistical significance determined using Student’s t test. *, P
Figure Legend Snippet: Aging is associated with decreased mTOR signaling within the BM microenvironment. (A) Representative immunoblot images demonstrating decreased expression of phospho-S6 and phospho-4EBP-1 in BM cells of aged mice compared with those of young mice. (B) Densitometry-based quantification of indicated proteins in the BM of aged mice compared with young mice ( n = 6 mice per cohort). Expression of Actb was used for normalization. Data represent combined analysis of two independent experiments. (C and D) Quantification of mean fluorescent intensity (MFI) of phospho-mTOR (Ser2448), phospho-AKT (Ser473), and phospho-S6 (Ser235/236) by Phosphoflow cytometry in Lin − CD45 + HSPCs (C) and Lin − CD45 − CD31 + VECAD + BMECs (D; n = 5 mice per cohort). (E) Representative immunoblot images demonstrating decreased expression of phospho-S6 and phospho-4EBP-1 in BM cells of aged mice treated with Rapamycin. (F) Densitometry-based quantification of indicated proteins in the BM of aged mice treated with Rapamycin compared with aged control mice ( n = 3 mice per cohort). Expression of Actb was used for normalization. (G–I) Analysis of wild-type cre - ( n = 3) and C dh 5-creERT2 + ( n = 3) mice revealed no significant differences in BM cellularity (G), HSC frequency (H), and peripheral blood lineage composition (I) indicating that endothelial-specific expression of the creERT2 transgene does not affect hematopoiesis. (J–L) Hematopoietic analysis of heterozygote mTOR fl/+ creERT2 + ( n = 5) and mTOR (ECKO) ( n = 5) demonstrated that unlike mTOR (ECKO) mice, the littermate heterozygote mTOR fl/+ creERT2 + mice do not manifest increased BM cellularity (J), increased HSC frequency (K), and myeloid-skewed peripheral blood lineage composition (L). All mice used in G–L were administered 200 mg/kg tamoxifen via intraperitoneal injection at a concentration of 30 mg/ml in sunflower oil on consecutive 3 d, followed by 3 d off, and an additional 3 d of injection. Note that the same regimen induces HSPC aging phenotypes in homozygote mTOR fl/fl cre-ERT2 + ( Fig. 3 and Fig. 4 ), indicating that loss of both alleles of endothelial mTOR is essential to induce HSPC aging phenotypes. Error bars represent mean ± SEM. Statistical significance determined using Student’s t test. *, P

Techniques Used: Expressing, Mouse Assay, Cytometry, Injection, Concentration Assay

10) Product Images from "Loss of Epithelial p53 and αv Integrin Cooperate through Akt to Induce Squamous Cell Carcinoma yet Prevent Remodeling of the Tumor Microenvironment"

Article Title: Loss of Epithelial p53 and αv Integrin Cooperate through Akt to Induce Squamous Cell Carcinoma yet Prevent Remodeling of the Tumor Microenvironment

Journal: Oncogene

doi: 10.1038/onc.2013.585

Defective angiogenesis in SCCs induced by co-deletion of epithelial αv and p53 ( a ) Immunohistochemical staining for α-SMA in αv + /p53 − , αv − /p53 + , and αv − /p53 − SCCs. ( b ) Quantification of the staining shown in panel a . ( c ) Double immunofluorescence for CD31 (endothelial cells, in green) and K14 (epithelial tumor cells, in red) in the indicated tumors. Arrows point to vessels that contain well-defined lumens. ( d ) Quantification of the area covered by endothelial cells in the tumors. * p
Figure Legend Snippet: Defective angiogenesis in SCCs induced by co-deletion of epithelial αv and p53 ( a ) Immunohistochemical staining for α-SMA in αv + /p53 − , αv − /p53 + , and αv − /p53 − SCCs. ( b ) Quantification of the staining shown in panel a . ( c ) Double immunofluorescence for CD31 (endothelial cells, in green) and K14 (epithelial tumor cells, in red) in the indicated tumors. Arrows point to vessels that contain well-defined lumens. ( d ) Quantification of the area covered by endothelial cells in the tumors. * p

Techniques Used: Immunohistochemistry, Staining, Immunofluorescence

11) Product Images from "Loss of Renal Peritubular Capillaries in Hypertensive Patients is Detectable by Urinary Endothelial Microparticle Levels"

Article Title: Loss of Renal Peritubular Capillaries in Hypertensive Patients is Detectable by Urinary Endothelial Microparticle Levels

Journal: Hypertension (Dallas, Tex. : 1979)

doi: 10.1161/HYPERTENSIONAHA.118.11766

Levels of exosomes in urine of hypertensive patients. (A) There were no differences among the groups in percent of urinary PL-VAP + /CD31 + , PL-VAP + /CD144 + , and PL-VAP + /CD31 + /CD144 + exosomes. (B) PTC-EMPs were identified using flow cytometry as PL-VAP + /CD31 − /CD144 − as shown in representative fluorescent images. Scale bar =20 µm. (C) Renal vein and systemic levels of PL-VAP + /CD31 − /CD144 − EMPs were not different among the groups, whereas their urinary levels were elevated in both EH and RVH compared to HVs (p
Figure Legend Snippet: Levels of exosomes in urine of hypertensive patients. (A) There were no differences among the groups in percent of urinary PL-VAP + /CD31 + , PL-VAP + /CD144 + , and PL-VAP + /CD31 + /CD144 + exosomes. (B) PTC-EMPs were identified using flow cytometry as PL-VAP + /CD31 − /CD144 − as shown in representative fluorescent images. Scale bar =20 µm. (C) Renal vein and systemic levels of PL-VAP + /CD31 − /CD144 − EMPs were not different among the groups, whereas their urinary levels were elevated in both EH and RVH compared to HVs (p

Techniques Used: Flow Cytometry, Cytometry

12) Product Images from "USP12 downregulation orchestrates a protumourigenic microenvironment and enhances lung tumour resistance to PD-1 blockade"

Article Title: USP12 downregulation orchestrates a protumourigenic microenvironment and enhances lung tumour resistance to PD-1 blockade

Journal: Nature Communications

doi: 10.1038/s41467-021-25032-5

USP12 modulates immune cell composition and activation in tumour microenvironment. a – d Flow cytometric analysis of the proportion of TAMs ( a ), PD-L1 expression on TAMs ( b ), PD-L1 expression on CD45 − cells ( c ), and the proportion of CD31 + CD45 − cells ( d ) in LLC tumours (mean ± SEM, n = 8 per group). 2-tailed unpaired t -test. e Flow cytometric analysis of the proportion of TAMs and PD-L1 expression on TAMs in indicated 889-S1 tumours (mean ± SEM, n = 4−5 per group). One-way ANOVA followed by Tukey’s HSD test. f Quantification analysis of CD31 immunostaining of indicated 889-S1 tumours (mean ± SEM, n = 20 fields of view from > 3 mice). Kruskal-Wallis test. g The proportion of TNF-α + IFN-γ + cells gated on CD8 and CD4 T cells in indicated 889-S1 tumours (mean ± SEM, n = 4–5 per group). One-way ANOVA followed by Tukey’s HSD test. h Boxplot showing the xCell scores for macrophages in USP12_low (bottom 25%) and USP12_high (top 25%) NSCLC samples. The centre mark represents the median, and whiskers show minimum/maximum values. Sample sizes for each group are given in parentheses. 2-tailed unpaired t -test. i Heat map of M2 myeloid cell markers and cytokines that were negatively correlated with USP12 ( P
Figure Legend Snippet: USP12 modulates immune cell composition and activation in tumour microenvironment. a – d Flow cytometric analysis of the proportion of TAMs ( a ), PD-L1 expression on TAMs ( b ), PD-L1 expression on CD45 − cells ( c ), and the proportion of CD31 + CD45 − cells ( d ) in LLC tumours (mean ± SEM, n = 8 per group). 2-tailed unpaired t -test. e Flow cytometric analysis of the proportion of TAMs and PD-L1 expression on TAMs in indicated 889-S1 tumours (mean ± SEM, n = 4−5 per group). One-way ANOVA followed by Tukey’s HSD test. f Quantification analysis of CD31 immunostaining of indicated 889-S1 tumours (mean ± SEM, n = 20 fields of view from > 3 mice). Kruskal-Wallis test. g The proportion of TNF-α + IFN-γ + cells gated on CD8 and CD4 T cells in indicated 889-S1 tumours (mean ± SEM, n = 4–5 per group). One-way ANOVA followed by Tukey’s HSD test. h Boxplot showing the xCell scores for macrophages in USP12_low (bottom 25%) and USP12_high (top 25%) NSCLC samples. The centre mark represents the median, and whiskers show minimum/maximum values. Sample sizes for each group are given in parentheses. 2-tailed unpaired t -test. i Heat map of M2 myeloid cell markers and cytokines that were negatively correlated with USP12 ( P

Techniques Used: Activation Assay, Expressing, Immunostaining, Mouse Assay

13) Product Images from "Reprogramming NK cells and macrophages via combined antibody and cytokine therapy primes tumors for elimination by checkpoint blockade"

Article Title: Reprogramming NK cells and macrophages via combined antibody and cytokine therapy primes tumors for elimination by checkpoint blockade

Journal: Cell reports

doi: 10.1016/j.celrep.2021.110021

1X AIP treatment induces tumor blood vessel normalization Mice bearing B16F10 tumors (n = 5–6 animals/group, pooled from two independent experiments) were left UT, treated with ICB alone, or treated with 1X AIP + ICB as in Figure 1G . Three days later, 70 kDa fluorescent dextran and Hoechst dye were injected i.v., and animals were sacrificed 10 min later for tumor harvest and immunohistochemistry. (A) Representative histological sections of UT, AIP-treated, and ICB-treated tumors. Green, CD31 staining; pink, dextran; blue, Hoechst staining. Scale bars are 100 μm and 500 μm for the first column of and the rest of the images, respectively. (B) Quantification of the radius of CD31 + blood vessels from CUBIC-R-cleared B16F10 tumors 3 days post AIP and ICB treatment or left UT. (C and D) Quantifications of CD31 + dextran + cells (C) and the fraction of Hoechst + areas (D) shown in (A). (E) Quantification of the ratio of endothelial cells to pericytes by flow cytometry from UT, AIP-treated, or ICB-treated B16F10-bearing mice. (F) Quantification of the hypoxia levels in tumors marked using the hypoxia-detecting molecule EF5 in mice treated as described in (D). (G) Quantification of total number and activated intratumoral endothelial cells in mice treated as described in (D). *p
Figure Legend Snippet: 1X AIP treatment induces tumor blood vessel normalization Mice bearing B16F10 tumors (n = 5–6 animals/group, pooled from two independent experiments) were left UT, treated with ICB alone, or treated with 1X AIP + ICB as in Figure 1G . Three days later, 70 kDa fluorescent dextran and Hoechst dye were injected i.v., and animals were sacrificed 10 min later for tumor harvest and immunohistochemistry. (A) Representative histological sections of UT, AIP-treated, and ICB-treated tumors. Green, CD31 staining; pink, dextran; blue, Hoechst staining. Scale bars are 100 μm and 500 μm for the first column of and the rest of the images, respectively. (B) Quantification of the radius of CD31 + blood vessels from CUBIC-R-cleared B16F10 tumors 3 days post AIP and ICB treatment or left UT. (C and D) Quantifications of CD31 + dextran + cells (C) and the fraction of Hoechst + areas (D) shown in (A). (E) Quantification of the ratio of endothelial cells to pericytes by flow cytometry from UT, AIP-treated, or ICB-treated B16F10-bearing mice. (F) Quantification of the hypoxia levels in tumors marked using the hypoxia-detecting molecule EF5 in mice treated as described in (D). (G) Quantification of total number and activated intratumoral endothelial cells in mice treated as described in (D). *p

Techniques Used: Mouse Assay, Injection, Immunohistochemistry, Staining, Flow Cytometry

14) Product Images from "EXTRACELLULAR VESICLES GENERATED BY PLACENTAL TISSUES EX VIVO: A TRANSPORT SYSTEM FOR IMMUNE MEDIATORS AND GROWTH FACTORS"

Article Title: EXTRACELLULAR VESICLES GENERATED BY PLACENTAL TISSUES EX VIVO: A TRANSPORT SYSTEM FOR IMMUNE MEDIATORS AND GROWTH FACTORS

Journal: American journal of reproductive immunology (New York, N.Y. : 1989)

doi: 10.1111/aji.12860

Distribution of cytokines between the surface and inner volume of EVs from placental villous tissues Distribution between encapsulated and surface cytokines is shown for placental villous cultures. (a) Total EVs isolated by Exoquick™ (b) anti-PLAP MNP-captured EVs; (c) anti-CD31 MNP-captured EVs; (d) anti-HLA-G MNP-captured EVs. Free and EV-associated cytokines are expressed as percent of total (Mean ± SEM, n=5). Blue bars: surface-associated cytokines, red: EV-encapsulated. Multiplexed bead assay measurements on samples collected at day 4 (cumulative amount for days 1–4 of culture).
Figure Legend Snippet: Distribution of cytokines between the surface and inner volume of EVs from placental villous tissues Distribution between encapsulated and surface cytokines is shown for placental villous cultures. (a) Total EVs isolated by Exoquick™ (b) anti-PLAP MNP-captured EVs; (c) anti-CD31 MNP-captured EVs; (d) anti-HLA-G MNP-captured EVs. Free and EV-associated cytokines are expressed as percent of total (Mean ± SEM, n=5). Blue bars: surface-associated cytokines, red: EV-encapsulated. Multiplexed bead assay measurements on samples collected at day 4 (cumulative amount for days 1–4 of culture).

Techniques Used: Isolation

Distribution of growth factors between the surface and inner volume of EVs from placental villous tissues Distribution between encapsulated and surface growth factors is shown for placental villous cultures. (a) Total EVs isolated by Exoquick™; (b) anti-PLAP MNP-captured EVs; (c) anti-CD31 MNP-captured EVs; (d) anti-HLA-G MNP- captured EVs. Free and EV-associated growth factors are expressed as percent of total (Mean ± SEM, n=5). Blue bars: surface-associated growth factors, red: EV-encapsulated. Multiplexed bead assay measurements on samples collected at day 4 (cumulative amount for days 1–4 of culture).
Figure Legend Snippet: Distribution of growth factors between the surface and inner volume of EVs from placental villous tissues Distribution between encapsulated and surface growth factors is shown for placental villous cultures. (a) Total EVs isolated by Exoquick™; (b) anti-PLAP MNP-captured EVs; (c) anti-CD31 MNP-captured EVs; (d) anti-HLA-G MNP- captured EVs. Free and EV-associated growth factors are expressed as percent of total (Mean ± SEM, n=5). Blue bars: surface-associated growth factors, red: EV-encapsulated. Multiplexed bead assay measurements on samples collected at day 4 (cumulative amount for days 1–4 of culture).

Techniques Used: Isolation

15) Product Images from "Metformin treatment status and abdominal aortic aneurysm disease progression"

Article Title: Metformin treatment status and abdominal aortic aneurysm disease progression

Journal: Journal of vascular surgery

doi: 10.1016/j.jvs.2016.02.020

Representative histology from metformin- and vehicle-treated, PPE-infused mice Aortic frozen sections were prepared 14 days following AAA creation. Elastin integrity was assessed via the EVG stain. Mural macrophages and angiogenesis were stained with mAbs against CD68 and CD31, respectively, via immunohistochemical staining. Representative images shown. n=7 and 10 mice in vehicle and metformin groups, respectively. Original magnification: 200X.
Figure Legend Snippet: Representative histology from metformin- and vehicle-treated, PPE-infused mice Aortic frozen sections were prepared 14 days following AAA creation. Elastin integrity was assessed via the EVG stain. Mural macrophages and angiogenesis were stained with mAbs against CD68 and CD31, respectively, via immunohistochemical staining. Representative images shown. n=7 and 10 mice in vehicle and metformin groups, respectively. Original magnification: 200X.

Techniques Used: Mouse Assay, Staining, Immunohistochemistry

Effects of metformin treatment on medial elastin and SMC integrity and mural inflammation in experimental AAA Aortic frozen sections from metformin- (n=10) and vehicle- (n=7) treated mice 14 days following PPE infusion were stained for elastin (EVG), SMC (SMC α-actin) and leukocyte subsets (CD68 for macrophages, CD4 for CD4 T cells, CD8 for CD8 T cells, B220 for B cells, CD31 for angiogenesis). Elastin degradation, SMC depletion and mural macrophage infiltration were graded as score I (mild) to score IV (severe). Mural CD4 T cells, CD8 T cells, B cells and angiogenesis were quantified as positive cells or blood vessels per ACS. Non-parametric Mann-Whitney test, *P
Figure Legend Snippet: Effects of metformin treatment on medial elastin and SMC integrity and mural inflammation in experimental AAA Aortic frozen sections from metformin- (n=10) and vehicle- (n=7) treated mice 14 days following PPE infusion were stained for elastin (EVG), SMC (SMC α-actin) and leukocyte subsets (CD68 for macrophages, CD4 for CD4 T cells, CD8 for CD8 T cells, B220 for B cells, CD31 for angiogenesis). Elastin degradation, SMC depletion and mural macrophage infiltration were graded as score I (mild) to score IV (severe). Mural CD4 T cells, CD8 T cells, B cells and angiogenesis were quantified as positive cells or blood vessels per ACS. Non-parametric Mann-Whitney test, *P

Techniques Used: Mouse Assay, Staining, MANN-WHITNEY

16) Product Images from "Myh11+ microvascular mural cells and derived mesenchymal stem cells promote retinal fibrosis"

Article Title: Myh11+ microvascular mural cells and derived mesenchymal stem cells promote retinal fibrosis

Journal: Scientific Reports

doi: 10.1038/s41598-020-72875-x

Adipose-derived, lineage-marked Myh11+ mural cells give rise to mesenchymal stem cells (MSCs) during adaptation and growth in vitro . ( A ) Immunostained epididymal adipose tissue from Myh11 -eYFP mice indicated eYFP+ (green) lineage marker is expressed in microvascular smooth muscle cells (vSMCs) (arrowhead) and microvascular pericytes (PCs) (asterisk) along lectin + blood vessels. Scale bar, 50 µm. ( B ) Immunostained adipose tissue revealed vSMC “tire tread” pattern on larger arterioles. Scale bar, 25 µm. ( C ) PCs are wrapped around adipose capillary microvasculature. Scale bar, 10 µm. ( D ) Flow cytometry analysis showed adipose Myh11+ mural cells collected from the SVF have relatively low endogenous expression of CD73, CD90, CD105, and CD146, however, after isolation via fluorescence activated cell-sorting (FACS), cultured, passage 3–5 Myh11+ mural cells significantly increased expression of CD73, CD90, CD105, and CD146 in vitro (three independent flow analyses per panel). ( E ) Graphical representation of flow cytometry analysis demonstrated significant increase of MSC surface antigens in Myh11+ mural cells after isolation from the SVF and cultured in vitro . ( F ) Flow cytometry analysis also revealed FAC-sorted and cultured passage 3–5 Myh11+ mural cells lacked expression for hematopoetic, endothelial, and macrophage markers CD11b, CD19, CD34, CD31, and CD45 (three independent flow analyses per panel). ( G , H ) Protein and genetic analysis of passage 2 Myh11+ mural cells when cultured in adipogeneic, chondrogenic, or osteogenic media for 14 days. ( G ) Increase in FABP4, Collagen II, and Osteopontin was observed by immunohistochemistry in Myh11+ mural cells undergoing tri-differentiation. Scale bar, 50 µm. ( H ) qPCR showed mRNA expression of protein markers and transcription factors involved in adipogenesis, chondrogenesis, and osteogenesis were significantly upregulated in Myh11+ mural cells following tri-differentiation (n = 3 biological replicates). Relative expression is normalized to GAPDH expression in each sample. Results are represented as mean ± standard error of mean (SEM). Data were analyzed using multiple unpaired t tests followed by the Holm–Sidak post-hoc comparisons to correct for multiple comparisons ( E ), or a ratio paired t-test ( H ). *p
Figure Legend Snippet: Adipose-derived, lineage-marked Myh11+ mural cells give rise to mesenchymal stem cells (MSCs) during adaptation and growth in vitro . ( A ) Immunostained epididymal adipose tissue from Myh11 -eYFP mice indicated eYFP+ (green) lineage marker is expressed in microvascular smooth muscle cells (vSMCs) (arrowhead) and microvascular pericytes (PCs) (asterisk) along lectin + blood vessels. Scale bar, 50 µm. ( B ) Immunostained adipose tissue revealed vSMC “tire tread” pattern on larger arterioles. Scale bar, 25 µm. ( C ) PCs are wrapped around adipose capillary microvasculature. Scale bar, 10 µm. ( D ) Flow cytometry analysis showed adipose Myh11+ mural cells collected from the SVF have relatively low endogenous expression of CD73, CD90, CD105, and CD146, however, after isolation via fluorescence activated cell-sorting (FACS), cultured, passage 3–5 Myh11+ mural cells significantly increased expression of CD73, CD90, CD105, and CD146 in vitro (three independent flow analyses per panel). ( E ) Graphical representation of flow cytometry analysis demonstrated significant increase of MSC surface antigens in Myh11+ mural cells after isolation from the SVF and cultured in vitro . ( F ) Flow cytometry analysis also revealed FAC-sorted and cultured passage 3–5 Myh11+ mural cells lacked expression for hematopoetic, endothelial, and macrophage markers CD11b, CD19, CD34, CD31, and CD45 (three independent flow analyses per panel). ( G , H ) Protein and genetic analysis of passage 2 Myh11+ mural cells when cultured in adipogeneic, chondrogenic, or osteogenic media for 14 days. ( G ) Increase in FABP4, Collagen II, and Osteopontin was observed by immunohistochemistry in Myh11+ mural cells undergoing tri-differentiation. Scale bar, 50 µm. ( H ) qPCR showed mRNA expression of protein markers and transcription factors involved in adipogenesis, chondrogenesis, and osteogenesis were significantly upregulated in Myh11+ mural cells following tri-differentiation (n = 3 biological replicates). Relative expression is normalized to GAPDH expression in each sample. Results are represented as mean ± standard error of mean (SEM). Data were analyzed using multiple unpaired t tests followed by the Holm–Sidak post-hoc comparisons to correct for multiple comparisons ( E ), or a ratio paired t-test ( H ). *p

Techniques Used: Derivative Assay, In Vitro, Mouse Assay, Marker, Flow Cytometry, Expressing, Isolation, Fluorescence, FACS, Cell Culture, Immunohistochemistry, Real-time Polymerase Chain Reaction

Within the murine OIR model, intravitreal injected Myh11-derived MSCs in the vitreous gel exhibit a myofibroblast phenotype, while endogenous, retinal Myh11+ mural cells remain in a perivascular position. ( A ) Immunostained Myh11-derived MSCs lacked expression of Col-IV in vitro, however, ( B ) immunostained retinas revealed intravitreal injected Myh11-derived MSCs expressed Col-IV in the vitreous gel, which formed a dense, fibrotic pre-retinal membrane in murine OIR eyes. Scale bars, 200 µm. ( C ) Intravitreal injected passage 3–5 Myh11-derived MSCs expressed αSMA+ stress fibers and Col-IV, and ( D ) Myh11-derived MSCs have reduced expression of Myh11 following injection (arrow) compared to the endogenous Myh11 expressed in retinal mural cells. DAPI stained nuclei of underlying retinal ganglion cells in addition to injected MSCs. Scale bars, 100 µm. ( E ) Experimental design where tamoxifen is delivered postnatal day 1–3 Myh11- tdTomato mice to induce expression of tdTomato in Myh11+ mural cells. Induced mice are then exposed to hyperoxia from postnatal day 7–12 to cause OIR injury, with retinas harvested at P17 to determine cell fate of endogenous, retinal Myh11+ mural cells. ( F ) At P17, endogenous, retinal Myh11+ mural cells resided on Col-IV+/CD31+ vessels, with αSMA expression higher in vSMCs (arrow) than PCs (asterisk). Scale bar, 100 µm. ( G ) Myh11+ mural cells remained on vessel with no vSMCs-PCs found off vessel. Scale bar, 100 µm. ( H ) Neither retinal vSMCs or PCs extended processes from CD31 tip cells (arrow) at the leading front of the regenerating retinal microvasculature. Scale bar, 25 µm. Immunohistochemistry images represent fields of view that were sampled based on the presence of eYFP and tdTomato expression within culture and tissue. Images are also representative of at least three biological replicates or animals.
Figure Legend Snippet: Within the murine OIR model, intravitreal injected Myh11-derived MSCs in the vitreous gel exhibit a myofibroblast phenotype, while endogenous, retinal Myh11+ mural cells remain in a perivascular position. ( A ) Immunostained Myh11-derived MSCs lacked expression of Col-IV in vitro, however, ( B ) immunostained retinas revealed intravitreal injected Myh11-derived MSCs expressed Col-IV in the vitreous gel, which formed a dense, fibrotic pre-retinal membrane in murine OIR eyes. Scale bars, 200 µm. ( C ) Intravitreal injected passage 3–5 Myh11-derived MSCs expressed αSMA+ stress fibers and Col-IV, and ( D ) Myh11-derived MSCs have reduced expression of Myh11 following injection (arrow) compared to the endogenous Myh11 expressed in retinal mural cells. DAPI stained nuclei of underlying retinal ganglion cells in addition to injected MSCs. Scale bars, 100 µm. ( E ) Experimental design where tamoxifen is delivered postnatal day 1–3 Myh11- tdTomato mice to induce expression of tdTomato in Myh11+ mural cells. Induced mice are then exposed to hyperoxia from postnatal day 7–12 to cause OIR injury, with retinas harvested at P17 to determine cell fate of endogenous, retinal Myh11+ mural cells. ( F ) At P17, endogenous, retinal Myh11+ mural cells resided on Col-IV+/CD31+ vessels, with αSMA expression higher in vSMCs (arrow) than PCs (asterisk). Scale bar, 100 µm. ( G ) Myh11+ mural cells remained on vessel with no vSMCs-PCs found off vessel. Scale bar, 100 µm. ( H ) Neither retinal vSMCs or PCs extended processes from CD31 tip cells (arrow) at the leading front of the regenerating retinal microvasculature. Scale bar, 25 µm. Immunohistochemistry images represent fields of view that were sampled based on the presence of eYFP and tdTomato expression within culture and tissue. Images are also representative of at least three biological replicates or animals.

Techniques Used: Injection, Derivative Assay, Expressing, In Vitro, Staining, Mouse Assay, Immunohistochemistry

Endogenous Myh11+ mural cells on the retinal microvasculature exhibit a myofibroblast phenotype after a chemical burn to the murine sclera. ( A ) Model demonstrating that silver-nitrate-burn injury to the sclera induced formation of retinal fibrotic scar tissue. ( B ) Immunostained, uninjured retinal tissue revealed Myh11+ mural cells, labeled by tdTomato, are found only on the CD31+ retinal microvasculature. Col-IV is expressed only in the basement membrane of the retinal microvasculature. Scale bar, 15 µm. ( C ) Immunostained retinas one-month post-burn injury showed multiple off-vessel Myh11+ mural cells (tdTomato+), which is indicated by the lack of overlap with the blood vessel endothelium marker CD31. ( D , E ) Off-vessel Myh11+ mural cells display αSMA+ stress fibers and are positive for Col-IV, Col-III, and F-actin as shown by fluorescently labeled phalloidin. Scale bar, 15 µm. Animals were tested beginning at 10–12 weeks of age. Immunohistochemistry images are representative of three uninjured and injured eyes of Myh11 -tdTomato mice. Field of view in injured eyes were selected based on the visual observation of off-vessel Myh11+ mural cells.
Figure Legend Snippet: Endogenous Myh11+ mural cells on the retinal microvasculature exhibit a myofibroblast phenotype after a chemical burn to the murine sclera. ( A ) Model demonstrating that silver-nitrate-burn injury to the sclera induced formation of retinal fibrotic scar tissue. ( B ) Immunostained, uninjured retinal tissue revealed Myh11+ mural cells, labeled by tdTomato, are found only on the CD31+ retinal microvasculature. Col-IV is expressed only in the basement membrane of the retinal microvasculature. Scale bar, 15 µm. ( C ) Immunostained retinas one-month post-burn injury showed multiple off-vessel Myh11+ mural cells (tdTomato+), which is indicated by the lack of overlap with the blood vessel endothelium marker CD31. ( D , E ) Off-vessel Myh11+ mural cells display αSMA+ stress fibers and are positive for Col-IV, Col-III, and F-actin as shown by fluorescently labeled phalloidin. Scale bar, 15 µm. Animals were tested beginning at 10–12 weeks of age. Immunohistochemistry images are representative of three uninjured and injured eyes of Myh11 -tdTomato mice. Field of view in injured eyes were selected based on the visual observation of off-vessel Myh11+ mural cells.

Techniques Used: Labeling, Marker, Immunohistochemistry, Mouse Assay

17) Product Images from "EXTRACELLULAR VESICLES GENERATED BY PLACENTAL TISSUES EX VIVO: A TRANSPORT SYSTEM FOR IMMUNE MEDIATORS AND GROWTH FACTORS"

Article Title: EXTRACELLULAR VESICLES GENERATED BY PLACENTAL TISSUES EX VIVO: A TRANSPORT SYSTEM FOR IMMUNE MEDIATORS AND GROWTH FACTORS

Journal: American journal of reproductive immunology (New York, N.Y. : 1989)

doi: 10.1111/aji.12860

Distribution of cytokines between the surface and inner volume of EVs from placental villous tissues Distribution between encapsulated and surface cytokines is shown for placental villous cultures. (a) Total EVs isolated by Exoquick™ (b) anti-PLAP MNP-captured EVs; (c) anti-CD31 MNP-captured EVs; (d) anti-HLA-G MNP-captured EVs. Free and EV-associated cytokines are expressed as percent of total (Mean ± SEM, n=5). Blue bars: surface-associated cytokines, red: EV-encapsulated. Multiplexed bead assay measurements on samples collected at day 4 (cumulative amount for days 1–4 of culture).
Figure Legend Snippet: Distribution of cytokines between the surface and inner volume of EVs from placental villous tissues Distribution between encapsulated and surface cytokines is shown for placental villous cultures. (a) Total EVs isolated by Exoquick™ (b) anti-PLAP MNP-captured EVs; (c) anti-CD31 MNP-captured EVs; (d) anti-HLA-G MNP-captured EVs. Free and EV-associated cytokines are expressed as percent of total (Mean ± SEM, n=5). Blue bars: surface-associated cytokines, red: EV-encapsulated. Multiplexed bead assay measurements on samples collected at day 4 (cumulative amount for days 1–4 of culture).

Techniques Used: Isolation

Distribution of growth factors between the surface and inner volume of EVs from placental villous tissues Distribution between encapsulated and surface growth factors is shown for placental villous cultures. (a) Total EVs isolated by Exoquick™; (b) anti-PLAP MNP-captured EVs; (c) anti-CD31 MNP-captured EVs; (d) anti-HLA-G MNP- captured EVs. Free and EV-associated growth factors are expressed as percent of total (Mean ± SEM, n=5). Blue bars: surface-associated growth factors, red: EV-encapsulated. Multiplexed bead assay measurements on samples collected at day 4 (cumulative amount for days 1–4 of culture).
Figure Legend Snippet: Distribution of growth factors between the surface and inner volume of EVs from placental villous tissues Distribution between encapsulated and surface growth factors is shown for placental villous cultures. (a) Total EVs isolated by Exoquick™; (b) anti-PLAP MNP-captured EVs; (c) anti-CD31 MNP-captured EVs; (d) anti-HLA-G MNP- captured EVs. Free and EV-associated growth factors are expressed as percent of total (Mean ± SEM, n=5). Blue bars: surface-associated growth factors, red: EV-encapsulated. Multiplexed bead assay measurements on samples collected at day 4 (cumulative amount for days 1–4 of culture).

Techniques Used: Isolation

18) Product Images from "EXTRACELLULAR VESICLES GENERATED BY PLACENTAL TISSUES EX VIVO: A TRANSPORT SYSTEM FOR IMMUNE MEDIATORS AND GROWTH FACTORS"

Article Title: EXTRACELLULAR VESICLES GENERATED BY PLACENTAL TISSUES EX VIVO: A TRANSPORT SYSTEM FOR IMMUNE MEDIATORS AND GROWTH FACTORS

Journal: American journal of reproductive immunology (New York, N.Y. : 1989)

doi: 10.1111/aji.12860

Distribution of cytokines between the surface and inner volume of EVs from placental villous tissues Distribution between encapsulated and surface cytokines is shown for placental villous cultures. (a) Total EVs isolated by Exoquick™ (b) anti-PLAP MNP-captured EVs; (c) anti-CD31 MNP-captured EVs; (d) anti-HLA-G MNP-captured EVs. Free and EV-associated cytokines are expressed as percent of total (Mean ± SEM, n=5). Blue bars: surface-associated cytokines, red: EV-encapsulated. Multiplexed bead assay measurements on samples collected at day 4 (cumulative amount for days 1–4 of culture).
Figure Legend Snippet: Distribution of cytokines between the surface and inner volume of EVs from placental villous tissues Distribution between encapsulated and surface cytokines is shown for placental villous cultures. (a) Total EVs isolated by Exoquick™ (b) anti-PLAP MNP-captured EVs; (c) anti-CD31 MNP-captured EVs; (d) anti-HLA-G MNP-captured EVs. Free and EV-associated cytokines are expressed as percent of total (Mean ± SEM, n=5). Blue bars: surface-associated cytokines, red: EV-encapsulated. Multiplexed bead assay measurements on samples collected at day 4 (cumulative amount for days 1–4 of culture).

Techniques Used: Isolation

Distribution of growth factors between the surface and inner volume of EVs from placental villous tissues Distribution between encapsulated and surface growth factors is shown for placental villous cultures. (a) Total EVs isolated by Exoquick™; (b) anti-PLAP MNP-captured EVs; (c) anti-CD31 MNP-captured EVs; (d) anti-HLA-G MNP- captured EVs. Free and EV-associated growth factors are expressed as percent of total (Mean ± SEM, n=5). Blue bars: surface-associated growth factors, red: EV-encapsulated. Multiplexed bead assay measurements on samples collected at day 4 (cumulative amount for days 1–4 of culture).
Figure Legend Snippet: Distribution of growth factors between the surface and inner volume of EVs from placental villous tissues Distribution between encapsulated and surface growth factors is shown for placental villous cultures. (a) Total EVs isolated by Exoquick™; (b) anti-PLAP MNP-captured EVs; (c) anti-CD31 MNP-captured EVs; (d) anti-HLA-G MNP- captured EVs. Free and EV-associated growth factors are expressed as percent of total (Mean ± SEM, n=5). Blue bars: surface-associated growth factors, red: EV-encapsulated. Multiplexed bead assay measurements on samples collected at day 4 (cumulative amount for days 1–4 of culture).

Techniques Used: Isolation

19) Product Images from "3D bioprinting of an implantable xeno-free vascularized human skin graft"

Article Title: 3D bioprinting of an implantable xeno-free vascularized human skin graft

Journal: bioRxiv

doi: 10.1101/2022.02.21.481363

Phenotypic characterization of HECFC-derived ECs cultured under xeno-free conditions compared to cells from the same donor cultured using EGM-2MV medium. (A) Live phase-contrast microscopy images of a HECFC-derived EC colony on fibronectin-coated plates after isolation from umbilical cord blood and at confluence. (B) The cumulative population doublings of ECs, determined by cell counting, for ECs cultured under xeno-free conditions vs. EGM2-Mv medium. (C) Formation of barriers to current flow over time as measured by transendothelial electrical resistance (TEER). (D) Barriers are equally disrupted in response to TNF-α (10 ng/mL). (E) Surface flow cytometry analysis demonstrating expression of CD31 and absence of CD45 expression. (F) Confocal microscopy exhibiting junctional ZO-1, VE-cadherin and claudin-5 staining and intracellular vWF staining in Weibel-Palade bodies. Scale bar: 100 μm. Representative of 3 independent donors.
Figure Legend Snippet: Phenotypic characterization of HECFC-derived ECs cultured under xeno-free conditions compared to cells from the same donor cultured using EGM-2MV medium. (A) Live phase-contrast microscopy images of a HECFC-derived EC colony on fibronectin-coated plates after isolation from umbilical cord blood and at confluence. (B) The cumulative population doublings of ECs, determined by cell counting, for ECs cultured under xeno-free conditions vs. EGM2-Mv medium. (C) Formation of barriers to current flow over time as measured by transendothelial electrical resistance (TEER). (D) Barriers are equally disrupted in response to TNF-α (10 ng/mL). (E) Surface flow cytometry analysis demonstrating expression of CD31 and absence of CD45 expression. (F) Confocal microscopy exhibiting junctional ZO-1, VE-cadherin and claudin-5 staining and intracellular vWF staining in Weibel-Palade bodies. Scale bar: 100 μm. Representative of 3 independent donors.

Techniques Used: Derivative Assay, Cell Culture, Microscopy, Isolation, Cell Counting, Flow Cytometry, Expressing, Confocal Microscopy, Staining

Phenotypic characterization of human placental PCs under xeno-free conditions. (A) Live phase-contrast microscopy images of a vessel fragment on a fibronectin-coated plate after isolation from placental tissue and atthe confluency state. (B) The cumulative population doublings of PCs cultured under xeno-free conditions is higher than in DMEM supplemented with 10% FBS. (C) Flow cytometry analysis of PCs cultured under xeno-free conditions confirmed the expression of PDGFR-α, NG2, CD90 and FAP. Furthermore, these cells lack expression of CD31 and CD45. (D) PCs showed positive expression of α-SMA and FAP, as confirmed by immunofluorescence staining. Scale bar: 100 μm. Representative of 3 independent donors.
Figure Legend Snippet: Phenotypic characterization of human placental PCs under xeno-free conditions. (A) Live phase-contrast microscopy images of a vessel fragment on a fibronectin-coated plate after isolation from placental tissue and atthe confluency state. (B) The cumulative population doublings of PCs cultured under xeno-free conditions is higher than in DMEM supplemented with 10% FBS. (C) Flow cytometry analysis of PCs cultured under xeno-free conditions confirmed the expression of PDGFR-α, NG2, CD90 and FAP. Furthermore, these cells lack expression of CD31 and CD45. (D) PCs showed positive expression of α-SMA and FAP, as confirmed by immunofluorescence staining. Scale bar: 100 μm. Representative of 3 independent donors.

Techniques Used: Microscopy, Isolation, Cell Culture, Flow Cytometry, Expressing, Immunofluorescence, Staining

Characterization of graft-derived and host-derived microvessel formation in xeno-free 3D bioprinted skin grafts with and without ECs and PCs 2-, 4- and 6-weeks post-engraftment onto immunodeficient mice. (A) Fluorescent mouse CD31 staining depicts higher degree of host angiogenesis (red) and the presence of perfused human EC-lined vessels (infused Ulex stain) in grafts containing ECs and PCs. (B) Human CD31 staining shows the presence of non-perfused human ECs (red) surrounded by PGA mesh and perfused human vessels (green). Nuclei are stained blue. Scale bar: 100um. Quantification of area of (C) infused ulex, (D) human CD31 and (E) mouse CD31 at 2-, 4-, and 6-weeks post-implantation. Data are shown for 3 independent experiments. (* indicates p
Figure Legend Snippet: Characterization of graft-derived and host-derived microvessel formation in xeno-free 3D bioprinted skin grafts with and without ECs and PCs 2-, 4- and 6-weeks post-engraftment onto immunodeficient mice. (A) Fluorescent mouse CD31 staining depicts higher degree of host angiogenesis (red) and the presence of perfused human EC-lined vessels (infused Ulex stain) in grafts containing ECs and PCs. (B) Human CD31 staining shows the presence of non-perfused human ECs (red) surrounded by PGA mesh and perfused human vessels (green). Nuclei are stained blue. Scale bar: 100um. Quantification of area of (C) infused ulex, (D) human CD31 and (E) mouse CD31 at 2-, 4-, and 6-weeks post-implantation. Data are shown for 3 independent experiments. (* indicates p

Techniques Used: Derivative Assay, Mouse Assay, Staining

20) Product Images from "Endothelial mTOR maintains hematopoiesis during aging"

Article Title: Endothelial mTOR maintains hematopoiesis during aging

Journal: bioRxiv

doi: 10.1101/2020.03.13.990911

Aging is associated with decreased mTOR signaling within the bone marrow microenvironment. (A) Representative immunoblot images demonstrating decreased expression of phospho-S6 and phsopho-4EBP-1 in BM cells of aged mice as compared to young mice. (B) Densitometry based quantification of indicated proteins in the BM of aged mice as compared to young mice (n=6 mice per cohort). Expression of Actb was used for normalization. Data represents combined analysis of 2 independent experiments. (C, D) Quantification of mean fluorescent intensity (MFI) of phospho-mTOR (Ser2448), phospho-AKT (Ser473) and phospho S6 (Ser235/236) by Phospho Flow cytometry in (C) Lin-CD45+ HSPCs and (D) Lin-CD45-CD31+VECAD+ BMECs (n=5 mice per cohort). (E) Representative immunoblot images demonstrating decreased expression of phospho-S6 and phsopho-4EBP-1 in BM cells of aged mice treated with Rapamycin. (F) Densitometry based quantification of indicated proteins in the BM of aged mice treated with Rapamycin as compared to aged control mice (n=3 mice per cohort). Expression of Actb was used for normalization. (G-I) Analysis of wild-type cre - (N=3) and CDH5-creERT2 + (N=3) mice revealed no significant differences in (G) BM cellularity, (H) HSC frequency, and (I) peripheral blood lineage composition indicating that endothelial-specific expression of cre-ERT2 transgene does not affect hematopoiesis. (J-L) Hematopoietic analysis of heterozygote mTOR fl/+ cre-ERT2+ (N=5) and mTOR (ECKO) (N=5) demonstrated that unlike mTOR (ECKO) mice, the littermate heterozygote mTOR fl/+ cre-ERT2+ mice do not manifest increased BM cellularity (J) , increased HSC frequency (K) , and myeloid-skewed peripheral blood lineage composition (L) . All mice utilized in (G-L) were administered 200 mg/kg tamoxifen via intraperitoneal injection at a concentration of 30 mg/mL in sunflower oil on three consecutive days, followed by three days off, and three additional days of injection. Note that the same regimen induces HSPC aging phenotypes in homozygote mTOR fl/fl cre-ERT2+ ( Figures 3 , 4 ), indicating that loss of both alleles of endothelial mTOR are essential to induce HSPC aging phenotypes. Error bars represent mean ± SEM. Statistical significance determined using Student’s t-test. *P
Figure Legend Snippet: Aging is associated with decreased mTOR signaling within the bone marrow microenvironment. (A) Representative immunoblot images demonstrating decreased expression of phospho-S6 and phsopho-4EBP-1 in BM cells of aged mice as compared to young mice. (B) Densitometry based quantification of indicated proteins in the BM of aged mice as compared to young mice (n=6 mice per cohort). Expression of Actb was used for normalization. Data represents combined analysis of 2 independent experiments. (C, D) Quantification of mean fluorescent intensity (MFI) of phospho-mTOR (Ser2448), phospho-AKT (Ser473) and phospho S6 (Ser235/236) by Phospho Flow cytometry in (C) Lin-CD45+ HSPCs and (D) Lin-CD45-CD31+VECAD+ BMECs (n=5 mice per cohort). (E) Representative immunoblot images demonstrating decreased expression of phospho-S6 and phsopho-4EBP-1 in BM cells of aged mice treated with Rapamycin. (F) Densitometry based quantification of indicated proteins in the BM of aged mice treated with Rapamycin as compared to aged control mice (n=3 mice per cohort). Expression of Actb was used for normalization. (G-I) Analysis of wild-type cre - (N=3) and CDH5-creERT2 + (N=3) mice revealed no significant differences in (G) BM cellularity, (H) HSC frequency, and (I) peripheral blood lineage composition indicating that endothelial-specific expression of cre-ERT2 transgene does not affect hematopoiesis. (J-L) Hematopoietic analysis of heterozygote mTOR fl/+ cre-ERT2+ (N=5) and mTOR (ECKO) (N=5) demonstrated that unlike mTOR (ECKO) mice, the littermate heterozygote mTOR fl/+ cre-ERT2+ mice do not manifest increased BM cellularity (J) , increased HSC frequency (K) , and myeloid-skewed peripheral blood lineage composition (L) . All mice utilized in (G-L) were administered 200 mg/kg tamoxifen via intraperitoneal injection at a concentration of 30 mg/mL in sunflower oil on three consecutive days, followed by three days off, and three additional days of injection. Note that the same regimen induces HSPC aging phenotypes in homozygote mTOR fl/fl cre-ERT2+ ( Figures 3 , 4 ), indicating that loss of both alleles of endothelial mTOR are essential to induce HSPC aging phenotypes. Error bars represent mean ± SEM. Statistical significance determined using Student’s t-test. *P

Techniques Used: Expressing, Mouse Assay, Flow Cytometry, Injection, Concentration Assay

21) Product Images from "Establishment of a translational endothelial cell model using directed differentiation of induced pluripotent stem cells from Cynomolgus monkey"

Article Title: Establishment of a translational endothelial cell model using directed differentiation of induced pluripotent stem cells from Cynomolgus monkey

Journal: Scientific Reports

doi: 10.1038/srep35830

Functional characterization of monkey IPSC-ECs. ( A ) Flow cytometry analysis of cIPSC-ECs at day 13 of differentiation. Cells express endothelial markers CD144, CD31 and CD309 and stain negative for hematopoietic lineage marker CD45. Unstained control samples are shown in grey. ( B ) Uptake of acetylated LDL by cIPSC-ECs. Overlay of phase contrast image and fluorescent channel shows incorporated acLDL (conjugated to Alexa488) in ECs. Scale bar: 50 μm. ( C ) Angiogenic potential of cIPSC-ECs demonstrated by tube formation assay. After 24 hours, cells form a network of tubular structures. Tube formation is inhibited in the presence of 10 μM sulforaphane or 2 μg/ml anti-VEGF antibody. Scale bars: 200 μm. Quantification of the inhibitory effect of anti-angiogenic molecules on the tubulogenesis of cIPSC-ECs (right panel). Columns show total tube length relative to control from three independent experiments. ( D ) Impedance-based monitoring of cIPSC-EC culture demonstrating the formation of a tight monolayer. One of 2 independent experiments performed is depicted (n = 4 technical replicates), error bar STD. ( E ) Response to proinflammatory stimuli. Twenty-four hours after stimulation with proinflammatory cytokines, cIPSC-ECs exhibit upregulated EC. activation marker ICAM1. Scale bars: 50 μm. Quantification of median intensity of ICAM1 staining (right panel). Columns show mean +/− STD of three independent experiments and data were analyzed using Student’s t-test.
Figure Legend Snippet: Functional characterization of monkey IPSC-ECs. ( A ) Flow cytometry analysis of cIPSC-ECs at day 13 of differentiation. Cells express endothelial markers CD144, CD31 and CD309 and stain negative for hematopoietic lineage marker CD45. Unstained control samples are shown in grey. ( B ) Uptake of acetylated LDL by cIPSC-ECs. Overlay of phase contrast image and fluorescent channel shows incorporated acLDL (conjugated to Alexa488) in ECs. Scale bar: 50 μm. ( C ) Angiogenic potential of cIPSC-ECs demonstrated by tube formation assay. After 24 hours, cells form a network of tubular structures. Tube formation is inhibited in the presence of 10 μM sulforaphane or 2 μg/ml anti-VEGF antibody. Scale bars: 200 μm. Quantification of the inhibitory effect of anti-angiogenic molecules on the tubulogenesis of cIPSC-ECs (right panel). Columns show total tube length relative to control from three independent experiments. ( D ) Impedance-based monitoring of cIPSC-EC culture demonstrating the formation of a tight monolayer. One of 2 independent experiments performed is depicted (n = 4 technical replicates), error bar STD. ( E ) Response to proinflammatory stimuli. Twenty-four hours after stimulation with proinflammatory cytokines, cIPSC-ECs exhibit upregulated EC. activation marker ICAM1. Scale bars: 50 μm. Quantification of median intensity of ICAM1 staining (right panel). Columns show mean +/− STD of three independent experiments and data were analyzed using Student’s t-test.

Techniques Used: Functional Assay, Flow Cytometry, Cytometry, Staining, Marker, Tube Formation Assay, Activation Assay

22) Product Images from "Testosterone is an endogenous regulator of BAFF and splenic B cell number"

Article Title: Testosterone is an endogenous regulator of BAFF and splenic B cell number

Journal: Nature Communications

doi: 10.1038/s41467-018-04408-0

Expansion of FRCs in testosterone/AR deficiency. a Quantification of smooth muscle a-actin (SMA)-stained area, expressed as percentage of peri-arteriolar lymphoid sheath (PALS) area in control ( Pgk-Cre + ) and general androgen receptor knockout (G-ARKO) male mice. n = 5–6/group. b Section of spleens from control and G-ARKO male mice, at the border of the B cell zone and PALS area. Red, SMA-positive fibroblastic reticular cells (FRC); turquoise, IgD-positive B cells. Scale bar, 30 µm. c Representative plots of PDPN + CD31 – CD45 – FRCs in the spleen from sham-operated and castrated (ORX) male mice 2 weeks after surgery. d Total number of CD45 – cells in spleens of castrated or sham-operated male mice. n = 15/group. e Frequency of FRC among stromal (CD45 – ) cells and f total FRCs per spleen in ORX and sham-operated male mice. Bars indicate means; circles represent individual mice.* P
Figure Legend Snippet: Expansion of FRCs in testosterone/AR deficiency. a Quantification of smooth muscle a-actin (SMA)-stained area, expressed as percentage of peri-arteriolar lymphoid sheath (PALS) area in control ( Pgk-Cre + ) and general androgen receptor knockout (G-ARKO) male mice. n = 5–6/group. b Section of spleens from control and G-ARKO male mice, at the border of the B cell zone and PALS area. Red, SMA-positive fibroblastic reticular cells (FRC); turquoise, IgD-positive B cells. Scale bar, 30 µm. c Representative plots of PDPN + CD31 – CD45 – FRCs in the spleen from sham-operated and castrated (ORX) male mice 2 weeks after surgery. d Total number of CD45 – cells in spleens of castrated or sham-operated male mice. n = 15/group. e Frequency of FRC among stromal (CD45 – ) cells and f total FRCs per spleen in ORX and sham-operated male mice. Bars indicate means; circles represent individual mice.* P

Techniques Used: Staining, Knock-Out, Mouse Assay

23) Product Images from "Using Imaging Flow Cytometry to Characterize Extracellular Vesicles Isolated from Cell Culture Media, Plasma or Urine"

Article Title: Using Imaging Flow Cytometry to Characterize Extracellular Vesicles Isolated from Cell Culture Media, Plasma or Urine

Journal: Bio-protocol

doi: 10.21769/BioProtoc.3420

A schematic showing flow cytometry gating strategy for PTC identification. Step 1: Scatterplot of side scatter (size) and intensity of Tag-It Violet (EVs) from all events collected, Draw gate (ROI) for Tag-It violet positive events and SSC low; Step 2: Histogram of PL-VAP antibody intensity of the EV+ population, draw gate (ROI) for PL-VAP positive events; Step 3: Scatterplot of CD31 and CD144 from PL-VAP+ population, most important gate (ROI) being CD31-/CD144- for PTC EV, however gates (ROIs) for identification of CD31-/CD144+, CD31+, CD144-, and CD31+/CD144+ events are also drawn.
Figure Legend Snippet: A schematic showing flow cytometry gating strategy for PTC identification. Step 1: Scatterplot of side scatter (size) and intensity of Tag-It Violet (EVs) from all events collected, Draw gate (ROI) for Tag-It violet positive events and SSC low; Step 2: Histogram of PL-VAP antibody intensity of the EV+ population, draw gate (ROI) for PL-VAP positive events; Step 3: Scatterplot of CD31 and CD144 from PL-VAP+ population, most important gate (ROI) being CD31-/CD144- for PTC EV, however gates (ROIs) for identification of CD31-/CD144+, CD31+, CD144-, and CD31+/CD144+ events are also drawn.

Techniques Used: Flow Cytometry

24) Product Images from "Endothelial mTOR maintains hematopoiesis during aging"

Article Title: Endothelial mTOR maintains hematopoiesis during aging

Journal: bioRxiv

doi: 10.1101/2020.03.13.990911

Aging is associated with decreased mTOR signaling within the bone marrow microenvironment. (A) Representative immunoblot images demonstrating decreased expression of phospho-S6 and phsopho-4EBP-1 in BM cells of aged mice as compared to young mice. (B) Densitometry based quantification of indicated proteins in the BM of aged mice as compared to young mice (n=6 mice per cohort). Expression of Actb was used for normalization. Data represents combined analysis of 2 independent experiments. (C, D) Quantification of mean fluorescent intensity (MFI) of phospho-mTOR (Ser2448), phospho-AKT (Ser473) and phospho S6 (Ser235/236) by Phospho Flow cytometry in (C) Lin-CD45+ HSPCs and (D) Lin-CD45-CD31+VECAD+ BMECs (n=5 mice per cohort). (E) Representative immunoblot images demonstrating decreased expression of phospho-S6 and phsopho-4EBP-1 in BM cells of aged mice treated with Rapamycin. (F) Densitometry based quantification of indicated proteins in the BM of aged mice treated with Rapamycin as compared to aged control mice (n=3 mice per cohort). Expression of Actb was used for normalization. (G-I) Analysis of wild-type cre - (N=3) and CDH5-creERT2 + (N=3) mice revealed no significant differences in (G) BM cellularity, (H) HSC frequency, and (I) peripheral blood lineage composition indicating that endothelial-specific expression of cre-ERT2 transgene does not affect hematopoiesis. (J-L) Hematopoietic analysis of heterozygote mTOR fl/+ cre-ERT2+ (N=5) and mTOR (ECKO) (N=5) demonstrated that unlike mTOR (ECKO) mice, the littermate heterozygote mTOR fl/+ cre-ERT2+ mice do not manifest increased BM cellularity (J) , increased HSC frequency (K) , and myeloid-skewed peripheral blood lineage composition (L) . All mice utilized in (G-L) were administered 200 mg/kg tamoxifen via intraperitoneal injection at a concentration of 30 mg/mL in sunflower oil on three consecutive days, followed by three days off, and three additional days of injection. Note that the same regimen induces HSPC aging phenotypes in homozygote mTOR fl/fl cre-ERT2+ ( Figures 3 , 4 ), indicating that loss of both alleles of endothelial mTOR are essential to induce HSPC aging phenotypes. Error bars represent mean ± SEM. Statistical significance determined using Student’s t-test. *P
Figure Legend Snippet: Aging is associated with decreased mTOR signaling within the bone marrow microenvironment. (A) Representative immunoblot images demonstrating decreased expression of phospho-S6 and phsopho-4EBP-1 in BM cells of aged mice as compared to young mice. (B) Densitometry based quantification of indicated proteins in the BM of aged mice as compared to young mice (n=6 mice per cohort). Expression of Actb was used for normalization. Data represents combined analysis of 2 independent experiments. (C, D) Quantification of mean fluorescent intensity (MFI) of phospho-mTOR (Ser2448), phospho-AKT (Ser473) and phospho S6 (Ser235/236) by Phospho Flow cytometry in (C) Lin-CD45+ HSPCs and (D) Lin-CD45-CD31+VECAD+ BMECs (n=5 mice per cohort). (E) Representative immunoblot images demonstrating decreased expression of phospho-S6 and phsopho-4EBP-1 in BM cells of aged mice treated with Rapamycin. (F) Densitometry based quantification of indicated proteins in the BM of aged mice treated with Rapamycin as compared to aged control mice (n=3 mice per cohort). Expression of Actb was used for normalization. (G-I) Analysis of wild-type cre - (N=3) and CDH5-creERT2 + (N=3) mice revealed no significant differences in (G) BM cellularity, (H) HSC frequency, and (I) peripheral blood lineage composition indicating that endothelial-specific expression of cre-ERT2 transgene does not affect hematopoiesis. (J-L) Hematopoietic analysis of heterozygote mTOR fl/+ cre-ERT2+ (N=5) and mTOR (ECKO) (N=5) demonstrated that unlike mTOR (ECKO) mice, the littermate heterozygote mTOR fl/+ cre-ERT2+ mice do not manifest increased BM cellularity (J) , increased HSC frequency (K) , and myeloid-skewed peripheral blood lineage composition (L) . All mice utilized in (G-L) were administered 200 mg/kg tamoxifen via intraperitoneal injection at a concentration of 30 mg/mL in sunflower oil on three consecutive days, followed by three days off, and three additional days of injection. Note that the same regimen induces HSPC aging phenotypes in homozygote mTOR fl/fl cre-ERT2+ ( Figures 3 , 4 ), indicating that loss of both alleles of endothelial mTOR are essential to induce HSPC aging phenotypes. Error bars represent mean ± SEM. Statistical significance determined using Student’s t-test. *P

Techniques Used: Expressing, Mouse Assay, Flow Cytometry, Injection, Concentration Assay

25) Product Images from "Identification of a common mesenchymal stromal progenitor for the adult haematopoietic niche"

Article Title: Identification of a common mesenchymal stromal progenitor for the adult haematopoietic niche

Journal: Nature Communications

doi: 10.1038/ncomms13095

CD 146 + and CD 166 + cells were marked by Col2.3-GFP or Osx1-Cre:GFP while Sca1 + cells were not. ( a – b ) FACS analysis of bone-disassociated stromal cells (CD45 − TER119 − CD31 − ) from ( a ) Col2.3-GFP or ( b ) Osx1-Cre:GFP mice. The fequency of the parent gate is displayed. ( c – d ) The frequency of Sca1+, CD146+, CD166+ and Sca1- in the GFP+ population for (mean±s.d.) ( c ) Col2.3-GFP and ( d ) Osx1-Cre:GFP obtained from FACS analysis ( n =3). ( e – f ) Sections of femurs from ( e ) Col2.3-GFP or ( f ) Osx1-Cre:GFP mice stained with antidodies to Sca1 or CD146.
Figure Legend Snippet: CD 146 + and CD 166 + cells were marked by Col2.3-GFP or Osx1-Cre:GFP while Sca1 + cells were not. ( a – b ) FACS analysis of bone-disassociated stromal cells (CD45 − TER119 − CD31 − ) from ( a ) Col2.3-GFP or ( b ) Osx1-Cre:GFP mice. The fequency of the parent gate is displayed. ( c – d ) The frequency of Sca1+, CD146+, CD166+ and Sca1- in the GFP+ population for (mean±s.d.) ( c ) Col2.3-GFP and ( d ) Osx1-Cre:GFP obtained from FACS analysis ( n =3). ( e – f ) Sections of femurs from ( e ) Col2.3-GFP or ( f ) Osx1-Cre:GFP mice stained with antidodies to Sca1 or CD146.

Techniques Used: FACS, Mouse Assay, Staining

Sca1 + progenitors contribute to BM stroma, while CD146 + and CD166 + progenitors form bones. ( a ) Direct transplants of GFP- labelled progenitors under the kidney capsule. ( b ) Bright-field and GFP images of GFP-labelled progenitors 1 month after transplant (far left and left). A representative cross-section of the graft site was stained with H E (right, green arrowhead points to bone) or GFP to identify the donor origin (far right, yellow arrowheads). ( c ) Co-tranplants of GFP-labelled adult progenitors with non-GFP fetal skeletal progenitors under the kidney capsule. ( d ) Bright-field and GFP images of GFP-labelled Sca1 + mixed with fetal skeletal progenitors 1 month after transplant. Donor-derived GFP + cells can be clearly identified (far left and left). Representative cross sections of the graft site stained with H E (right) or GFP ( far right) to identify the donor origin (yellow arrowheads). ( e – f ) Representative FACS analysis of graft of mixed GFP-labelled Sca1 + progenitors and non-GFP skeletal progenitors harvested 1 month after transplant. The per cent of live cells is displayed for each gate ( e ) FACS analysis of donor-derived endosteum associated progenitors (left) and marrow stromal cells (right). ( f ) FACS analysis for phenotypically defined CAR cells in control bone marrow, marrow of graft and kidney. ( g ) Percentage of GFP+ cells for each group; mean±s.d. ( n =3). Total stroma is the Ter119-CD45-CD31- population. ( h ) Histrogram for GFP to show the populations that were sorted for RNA isolation (far left). Relative expression of GFP, CD45 and CD31 in control cells (untransplanted kidney), GFP low gate cells and GFP high gate cells (* P
Figure Legend Snippet: Sca1 + progenitors contribute to BM stroma, while CD146 + and CD166 + progenitors form bones. ( a ) Direct transplants of GFP- labelled progenitors under the kidney capsule. ( b ) Bright-field and GFP images of GFP-labelled progenitors 1 month after transplant (far left and left). A representative cross-section of the graft site was stained with H E (right, green arrowhead points to bone) or GFP to identify the donor origin (far right, yellow arrowheads). ( c ) Co-tranplants of GFP-labelled adult progenitors with non-GFP fetal skeletal progenitors under the kidney capsule. ( d ) Bright-field and GFP images of GFP-labelled Sca1 + mixed with fetal skeletal progenitors 1 month after transplant. Donor-derived GFP + cells can be clearly identified (far left and left). Representative cross sections of the graft site stained with H E (right) or GFP ( far right) to identify the donor origin (yellow arrowheads). ( e – f ) Representative FACS analysis of graft of mixed GFP-labelled Sca1 + progenitors and non-GFP skeletal progenitors harvested 1 month after transplant. The per cent of live cells is displayed for each gate ( e ) FACS analysis of donor-derived endosteum associated progenitors (left) and marrow stromal cells (right). ( f ) FACS analysis for phenotypically defined CAR cells in control bone marrow, marrow of graft and kidney. ( g ) Percentage of GFP+ cells for each group; mean±s.d. ( n =3). Total stroma is the Ter119-CD45-CD31- population. ( h ) Histrogram for GFP to show the populations that were sorted for RNA isolation (far left). Relative expression of GFP, CD45 and CD31 in control cells (untransplanted kidney), GFP low gate cells and GFP high gate cells (* P

Techniques Used: Staining, Derivative Assay, FACS, Isolation, Expressing

Sca1 + progenitors hometo the BM after intravenous transplantation into irradiated mice. ( a ) Intravenous transfusion of GFP-lablled mesenchymal progenitors into irradiated mice (600 rad). ( b ) Representative cross sections of the tibia stained with H E (upper) or GFP (lower). Tibias were harvested 1 month after transplantation. Blue arrows point to GFP+ stromal cells ( c – d ) FACS analysis of the marrow of recipients injected with Sca1 + cells for ( c ) GFP expression or ( d ) phenotypes of CAR cells. ( e ) FACS analysis of donor-derived bone-disassociated progenitors (upper) and marrow stromal cells (lower). ( f ) Frequency of GFP+ cells in each group; mean±s.d. ( n =4). ( g ) Histrogram of GFP to show the population that was sorted for RNA isolation (far left). Relative expression of GFP, CD45 and CD31 in GFP- and GFP+ sorted cells from CD45 − Ter119 − CD31 − stromal cell gate (* P
Figure Legend Snippet: Sca1 + progenitors hometo the BM after intravenous transplantation into irradiated mice. ( a ) Intravenous transfusion of GFP-lablled mesenchymal progenitors into irradiated mice (600 rad). ( b ) Representative cross sections of the tibia stained with H E (upper) or GFP (lower). Tibias were harvested 1 month after transplantation. Blue arrows point to GFP+ stromal cells ( c – d ) FACS analysis of the marrow of recipients injected with Sca1 + cells for ( c ) GFP expression or ( d ) phenotypes of CAR cells. ( e ) FACS analysis of donor-derived bone-disassociated progenitors (upper) and marrow stromal cells (lower). ( f ) Frequency of GFP+ cells in each group; mean±s.d. ( n =4). ( g ) Histrogram of GFP to show the population that was sorted for RNA isolation (far left). Relative expression of GFP, CD45 and CD31 in GFP- and GFP+ sorted cells from CD45 − Ter119 − CD31 − stromal cell gate (* P

Techniques Used: Transplantation Assay, Irradiation, Mouse Assay, Staining, FACS, Injection, Expressing, Derivative Assay, Isolation

Phenotypic Identification of three bone-disassociated mesenchymal progenitors. ( a ) Representative FACS profiles of stromal cells (CD45 − Ter119 − CD31 − ) near the endosteum (bone-disassociated). They were separated based on their CD166, CD146 and Sca1 expression profiles. Numbers shown in the gate represent the percentage of total live cells. ( b ) Single-cell colony forming efficiency of BM stromal progenitors (*: P ≤0.05, three repeats of 60 cells each, student's t -test). ( c ) Sections of femur from Flk1-GFP mouse stained with antibodies against Sca1 and scanned with an iCys Research imaging cytometer. Nuclei were stained with Dapi. ( d ) Confocal maximum intensity projections of sections of femurs from Flk1-GFP mice stained with antibodies against Sca1 or CD146; scanned near the epiphyseal plate. ( e ) Confocal maximum intensity projections of sections of femur stained with antibodies against Sca1 or CD146 and laminin; scanned near epiphyseal plate. White arrows point to mesenchymal progenitors yellow arrows point to endothelial cells.
Figure Legend Snippet: Phenotypic Identification of three bone-disassociated mesenchymal progenitors. ( a ) Representative FACS profiles of stromal cells (CD45 − Ter119 − CD31 − ) near the endosteum (bone-disassociated). They were separated based on their CD166, CD146 and Sca1 expression profiles. Numbers shown in the gate represent the percentage of total live cells. ( b ) Single-cell colony forming efficiency of BM stromal progenitors (*: P ≤0.05, three repeats of 60 cells each, student's t -test). ( c ) Sections of femur from Flk1-GFP mouse stained with antibodies against Sca1 and scanned with an iCys Research imaging cytometer. Nuclei were stained with Dapi. ( d ) Confocal maximum intensity projections of sections of femurs from Flk1-GFP mice stained with antibodies against Sca1 or CD146; scanned near the epiphyseal plate. ( e ) Confocal maximum intensity projections of sections of femur stained with antibodies against Sca1 or CD146 and laminin; scanned near epiphyseal plate. White arrows point to mesenchymal progenitors yellow arrows point to endothelial cells.

Techniques Used: FACS, Expressing, Staining, Imaging, Cytometry, Mouse Assay

26) Product Images from "Myh11+ microvascular mural cells and derived mesenchymal stem cells promote retinal fibrosis"

Article Title: Myh11+ microvascular mural cells and derived mesenchymal stem cells promote retinal fibrosis

Journal: Scientific Reports

doi: 10.1038/s41598-020-72875-x

Adipose-derived, lineage-marked Myh11+ mural cells give rise to mesenchymal stem cells (MSCs) during adaptation and growth in vitro . ( A ) Immunostained epididymal adipose tissue from Myh11 -eYFP mice indicated eYFP+ (green) lineage marker is expressed in microvascular smooth muscle cells (vSMCs) (arrowhead) and microvascular pericytes (PCs) (asterisk) along lectin + blood vessels. Scale bar, 50 µm. ( B ) Immunostained adipose tissue revealed vSMC “tire tread” pattern on larger arterioles. Scale bar, 25 µm. ( C ) PCs are wrapped around adipose capillary microvasculature. Scale bar, 10 µm. ( D ) Flow cytometry analysis showed adipose Myh11+ mural cells collected from the SVF have relatively low endogenous expression of CD73, CD90, CD105, and CD146, however, after isolation via fluorescence activated cell-sorting (FACS), cultured, passage 3–5 Myh11+ mural cells significantly increased expression of CD73, CD90, CD105, and CD146 in vitro (three independent flow analyses per panel). ( E ) Graphical representation of flow cytometry analysis demonstrated significant increase of MSC surface antigens in Myh11+ mural cells after isolation from the SVF and cultured in vitro . ( F ) Flow cytometry analysis also revealed FAC-sorted and cultured passage 3–5 Myh11+ mural cells lacked expression for hematopoetic, endothelial, and macrophage markers CD11b, CD19, CD34, CD31, and CD45 (three independent flow analyses per panel). ( G , H ) Protein and genetic analysis of passage 2 Myh11+ mural cells when cultured in adipogeneic, chondrogenic, or osteogenic media for 14 days. ( G ) Increase in FABP4, Collagen II, and Osteopontin was observed by immunohistochemistry in Myh11+ mural cells undergoing tri-differentiation. Scale bar, 50 µm. ( H ) qPCR showed mRNA expression of protein markers and transcription factors involved in adipogenesis, chondrogenesis, and osteogenesis were significantly upregulated in Myh11+ mural cells following tri-differentiation (n = 3 biological replicates). Relative expression is normalized to GAPDH expression in each sample. Results are represented as mean ± standard error of mean (SEM). Data were analyzed using multiple unpaired t tests followed by the Holm–Sidak post-hoc comparisons to correct for multiple comparisons ( E ), or a ratio paired t-test ( H ). *p
Figure Legend Snippet: Adipose-derived, lineage-marked Myh11+ mural cells give rise to mesenchymal stem cells (MSCs) during adaptation and growth in vitro . ( A ) Immunostained epididymal adipose tissue from Myh11 -eYFP mice indicated eYFP+ (green) lineage marker is expressed in microvascular smooth muscle cells (vSMCs) (arrowhead) and microvascular pericytes (PCs) (asterisk) along lectin + blood vessels. Scale bar, 50 µm. ( B ) Immunostained adipose tissue revealed vSMC “tire tread” pattern on larger arterioles. Scale bar, 25 µm. ( C ) PCs are wrapped around adipose capillary microvasculature. Scale bar, 10 µm. ( D ) Flow cytometry analysis showed adipose Myh11+ mural cells collected from the SVF have relatively low endogenous expression of CD73, CD90, CD105, and CD146, however, after isolation via fluorescence activated cell-sorting (FACS), cultured, passage 3–5 Myh11+ mural cells significantly increased expression of CD73, CD90, CD105, and CD146 in vitro (three independent flow analyses per panel). ( E ) Graphical representation of flow cytometry analysis demonstrated significant increase of MSC surface antigens in Myh11+ mural cells after isolation from the SVF and cultured in vitro . ( F ) Flow cytometry analysis also revealed FAC-sorted and cultured passage 3–5 Myh11+ mural cells lacked expression for hematopoetic, endothelial, and macrophage markers CD11b, CD19, CD34, CD31, and CD45 (three independent flow analyses per panel). ( G , H ) Protein and genetic analysis of passage 2 Myh11+ mural cells when cultured in adipogeneic, chondrogenic, or osteogenic media for 14 days. ( G ) Increase in FABP4, Collagen II, and Osteopontin was observed by immunohistochemistry in Myh11+ mural cells undergoing tri-differentiation. Scale bar, 50 µm. ( H ) qPCR showed mRNA expression of protein markers and transcription factors involved in adipogenesis, chondrogenesis, and osteogenesis were significantly upregulated in Myh11+ mural cells following tri-differentiation (n = 3 biological replicates). Relative expression is normalized to GAPDH expression in each sample. Results are represented as mean ± standard error of mean (SEM). Data were analyzed using multiple unpaired t tests followed by the Holm–Sidak post-hoc comparisons to correct for multiple comparisons ( E ), or a ratio paired t-test ( H ). *p

Techniques Used: Derivative Assay, In Vitro, Mouse Assay, Marker, Flow Cytometry, Expressing, Isolation, Fluorescence, FACS, Cell Culture, Immunohistochemistry, Real-time Polymerase Chain Reaction

Within the murine OIR model, intravitreal injected Myh11-derived MSCs in the vitreous gel exhibit a myofibroblast phenotype, while endogenous, retinal Myh11+ mural cells remain in a perivascular position. ( A ) Immunostained Myh11-derived MSCs lacked expression of Col-IV in vitro, however, ( B ) immunostained retinas revealed intravitreal injected Myh11-derived MSCs expressed Col-IV in the vitreous gel, which formed a dense, fibrotic pre-retinal membrane in murine OIR eyes. Scale bars, 200 µm. ( C ) Intravitreal injected passage 3–5 Myh11-derived MSCs expressed αSMA+ stress fibers and Col-IV, and ( D ) Myh11-derived MSCs have reduced expression of Myh11 following injection (arrow) compared to the endogenous Myh11 expressed in retinal mural cells. DAPI stained nuclei of underlying retinal ganglion cells in addition to injected MSCs. Scale bars, 100 µm. ( E ) Experimental design where tamoxifen is delivered postnatal day 1–3 Myh11- tdTomato mice to induce expression of tdTomato in Myh11+ mural cells. Induced mice are then exposed to hyperoxia from postnatal day 7–12 to cause OIR injury, with retinas harvested at P17 to determine cell fate of endogenous, retinal Myh11+ mural cells. ( F ) At P17, endogenous, retinal Myh11+ mural cells resided on Col-IV+/CD31+ vessels, with αSMA expression higher in vSMCs (arrow) than PCs (asterisk). Scale bar, 100 µm. ( G ) Myh11+ mural cells remained on vessel with no vSMCs-PCs found off vessel. Scale bar, 100 µm. ( H ) Neither retinal vSMCs or PCs extended processes from CD31 tip cells (arrow) at the leading front of the regenerating retinal microvasculature. Scale bar, 25 µm. Immunohistochemistry images represent fields of view that were sampled based on the presence of eYFP and tdTomato expression within culture and tissue. Images are also representative of at least three biological replicates or animals.
Figure Legend Snippet: Within the murine OIR model, intravitreal injected Myh11-derived MSCs in the vitreous gel exhibit a myofibroblast phenotype, while endogenous, retinal Myh11+ mural cells remain in a perivascular position. ( A ) Immunostained Myh11-derived MSCs lacked expression of Col-IV in vitro, however, ( B ) immunostained retinas revealed intravitreal injected Myh11-derived MSCs expressed Col-IV in the vitreous gel, which formed a dense, fibrotic pre-retinal membrane in murine OIR eyes. Scale bars, 200 µm. ( C ) Intravitreal injected passage 3–5 Myh11-derived MSCs expressed αSMA+ stress fibers and Col-IV, and ( D ) Myh11-derived MSCs have reduced expression of Myh11 following injection (arrow) compared to the endogenous Myh11 expressed in retinal mural cells. DAPI stained nuclei of underlying retinal ganglion cells in addition to injected MSCs. Scale bars, 100 µm. ( E ) Experimental design where tamoxifen is delivered postnatal day 1–3 Myh11- tdTomato mice to induce expression of tdTomato in Myh11+ mural cells. Induced mice are then exposed to hyperoxia from postnatal day 7–12 to cause OIR injury, with retinas harvested at P17 to determine cell fate of endogenous, retinal Myh11+ mural cells. ( F ) At P17, endogenous, retinal Myh11+ mural cells resided on Col-IV+/CD31+ vessels, with αSMA expression higher in vSMCs (arrow) than PCs (asterisk). Scale bar, 100 µm. ( G ) Myh11+ mural cells remained on vessel with no vSMCs-PCs found off vessel. Scale bar, 100 µm. ( H ) Neither retinal vSMCs or PCs extended processes from CD31 tip cells (arrow) at the leading front of the regenerating retinal microvasculature. Scale bar, 25 µm. Immunohistochemistry images represent fields of view that were sampled based on the presence of eYFP and tdTomato expression within culture and tissue. Images are also representative of at least three biological replicates or animals.

Techniques Used: Injection, Derivative Assay, Expressing, In Vitro, Staining, Mouse Assay, Immunohistochemistry

Endogenous Myh11+ mural cells on the retinal microvasculature exhibit a myofibroblast phenotype after a chemical burn to the murine sclera. ( A ) Model demonstrating that silver-nitrate-burn injury to the sclera induced formation of retinal fibrotic scar tissue. ( B ) Immunostained, uninjured retinal tissue revealed Myh11+ mural cells, labeled by tdTomato, are found only on the CD31+ retinal microvasculature. Col-IV is expressed only in the basement membrane of the retinal microvasculature. Scale bar, 15 µm. ( C ) Immunostained retinas one-month post-burn injury showed multiple off-vessel Myh11+ mural cells (tdTomato+), which is indicated by the lack of overlap with the blood vessel endothelium marker CD31. ( D , E ) Off-vessel Myh11+ mural cells display αSMA+ stress fibers and are positive for Col-IV, Col-III, and F-actin as shown by fluorescently labeled phalloidin. Scale bar, 15 µm. Animals were tested beginning at 10–12 weeks of age. Immunohistochemistry images are representative of three uninjured and injured eyes of Myh11 -tdTomato mice. Field of view in injured eyes were selected based on the visual observation of off-vessel Myh11+ mural cells.
Figure Legend Snippet: Endogenous Myh11+ mural cells on the retinal microvasculature exhibit a myofibroblast phenotype after a chemical burn to the murine sclera. ( A ) Model demonstrating that silver-nitrate-burn injury to the sclera induced formation of retinal fibrotic scar tissue. ( B ) Immunostained, uninjured retinal tissue revealed Myh11+ mural cells, labeled by tdTomato, are found only on the CD31+ retinal microvasculature. Col-IV is expressed only in the basement membrane of the retinal microvasculature. Scale bar, 15 µm. ( C ) Immunostained retinas one-month post-burn injury showed multiple off-vessel Myh11+ mural cells (tdTomato+), which is indicated by the lack of overlap with the blood vessel endothelium marker CD31. ( D , E ) Off-vessel Myh11+ mural cells display αSMA+ stress fibers and are positive for Col-IV, Col-III, and F-actin as shown by fluorescently labeled phalloidin. Scale bar, 15 µm. Animals were tested beginning at 10–12 weeks of age. Immunohistochemistry images are representative of three uninjured and injured eyes of Myh11 -tdTomato mice. Field of view in injured eyes were selected based on the visual observation of off-vessel Myh11+ mural cells.

Techniques Used: Labeling, Marker, Immunohistochemistry, Mouse Assay

27) Product Images from "De novo and cell line models of human mammary cell transformation reveal an essential role for Yb-1 in multiple stages of human breast cancer"

Article Title: De novo and cell line models of human mammary cell transformation reveal an essential role for Yb-1 in multiple stages of human breast cancer

Journal: Cell Death and Differentiation

doi: 10.1038/s41418-021-00836-6

KRAS G12D upregulates YB-1 in de novo KRAS G12D -transformed normal human mammary cells. A Western blots showing YB-1 levels (relative to H3) in human BCs, LPs, LCs and stromal cells (SCs) isolated viably from three normal donors according to their differential surface expression of EPCAM and CD49f levels and absence of expression of CD45 and CD31 (top panel). B Representative views of YB-1 immunostaining of normal human mammary tissue (left) and 8-week tumours derived from KRAS G12D -transduced normal mammary cells isolated from the same three normal donors (right). Scale bar, 50 μm. Bar graph shows quantification of YB-1 expression. Data are from individual tumours (Normal, n = 8; de novo tumours, n = 8). C Representative FACS profile of a 4-week xenograft of inducible KRAS G12D -transduced human BCs obtained from mice maintained post-transplant on doxycyline-supplemented water (Dox) for just the first 2 weeks (left panel) or for the full 4 weeks (right panel) of the experiment. D KRAS mRNA levels measured in 4-week xenografts of inducible KRAS G12D -transduced cells obtained from mice maintained on Dox for the first 2 weeks only, or the full 4 weeks of the experiment, as shown. The dot plot shows KRAS relative expression compared to the No Dox control mice ( n = 2), as ΔΔCt values. E Representative views of YB-1 immunostaining of 4-week tumours derived from mice transplanted with cells transduced with a Dox-inducible KRAS G12D cDNA and maintained on Dox for the just the first 2 weeks or the full 4 weeks before the tumours were harvested for analysis ( N = 3 donors). Scale bar, 50 μm.
Figure Legend Snippet: KRAS G12D upregulates YB-1 in de novo KRAS G12D -transformed normal human mammary cells. A Western blots showing YB-1 levels (relative to H3) in human BCs, LPs, LCs and stromal cells (SCs) isolated viably from three normal donors according to their differential surface expression of EPCAM and CD49f levels and absence of expression of CD45 and CD31 (top panel). B Representative views of YB-1 immunostaining of normal human mammary tissue (left) and 8-week tumours derived from KRAS G12D -transduced normal mammary cells isolated from the same three normal donors (right). Scale bar, 50 μm. Bar graph shows quantification of YB-1 expression. Data are from individual tumours (Normal, n = 8; de novo tumours, n = 8). C Representative FACS profile of a 4-week xenograft of inducible KRAS G12D -transduced human BCs obtained from mice maintained post-transplant on doxycyline-supplemented water (Dox) for just the first 2 weeks (left panel) or for the full 4 weeks (right panel) of the experiment. D KRAS mRNA levels measured in 4-week xenografts of inducible KRAS G12D -transduced cells obtained from mice maintained on Dox for the first 2 weeks only, or the full 4 weeks of the experiment, as shown. The dot plot shows KRAS relative expression compared to the No Dox control mice ( n = 2), as ΔΔCt values. E Representative views of YB-1 immunostaining of 4-week tumours derived from mice transplanted with cells transduced with a Dox-inducible KRAS G12D cDNA and maintained on Dox for the just the first 2 weeks or the full 4 weeks before the tumours were harvested for analysis ( N = 3 donors). Scale bar, 50 μm.

Techniques Used: Transformation Assay, Western Blot, Isolation, Expressing, Immunostaining, Derivative Assay, FACS, Mouse Assay, Transduction

28) Product Images from "LRP5 in age-related changes in vascular and alveolar morphogenesis in the lung"

Article Title: LRP5 in age-related changes in vascular and alveolar morphogenesis in the lung

Journal: Aging (Albany NY)

doi: 10.18632/aging.101722

Age-dependent changes in vascular and alveolar structures in the mouse lungs. ( A ) H E-stained 2M and 24M old mouse lungs ( top , scale bar, 20 μm). Immunofluorescence micrographs showing CD31-positive blood vessels and AQP5-positive alveolar type-I epithelial cells ( 2nd ), CD31-positive blood vessels and SPB-positive alveolar type-II epithelial cells ( 3rd ), CD31-positive blood vessels and VEGFR2 expression ( 4th ), and CD31-positive blood vessels and Tie2 expression ( bottom ) in the 2M vs. 24M old mouse lungs (scale bar, 20 μm). ( B ) Graphs showing quantification of alveolar size (MLI, top ), alveolar number ( 2nd ), vessel diameter ( 3rd ), and area of ECs expressing VEGFR2 and Tie2 ( bottom ) in the 2M and 24M old mouse lungs (n=7, mean ± s.e.m., *, p
Figure Legend Snippet: Age-dependent changes in vascular and alveolar structures in the mouse lungs. ( A ) H E-stained 2M and 24M old mouse lungs ( top , scale bar, 20 μm). Immunofluorescence micrographs showing CD31-positive blood vessels and AQP5-positive alveolar type-I epithelial cells ( 2nd ), CD31-positive blood vessels and SPB-positive alveolar type-II epithelial cells ( 3rd ), CD31-positive blood vessels and VEGFR2 expression ( 4th ), and CD31-positive blood vessels and Tie2 expression ( bottom ) in the 2M vs. 24M old mouse lungs (scale bar, 20 μm). ( B ) Graphs showing quantification of alveolar size (MLI, top ), alveolar number ( 2nd ), vessel diameter ( 3rd ), and area of ECs expressing VEGFR2 and Tie2 ( bottom ) in the 2M and 24M old mouse lungs (n=7, mean ± s.e.m., *, p

Techniques Used: Staining, Immunofluorescence, Expressing

LRP5 mediates age-dependent decline in vascular and alveolar epithelial morphogenesis in the gel implanted on the mouse lungs. (A) Immunofluorescence micrographs showing CD31-positive blood vessels and AQP5-positive alveolar type-I epithelial cells ( top ), CD31-positive blood vessels and SPB-positive alveolar type-II epithelial cells ( 2nd ), CD31-positive blood vessels and VEGFR2 expression ( 3rd ), and CD31-positive blood vessels and Tie2 expression ( bottom ) in the fibrin gel implanted on the 2M vs. 24M old mouse lungs or in combination with LRP5 overexpression for 7 days (scale bar, 20 μm). ( B) Graphs showing quantification of CD31-positive blood vessel numbers ( left ), area of ECs expressing VEGFR2 ( middle ) and Tie2 ( right ) in the gel implanted on the 2M vs. 24M old mouse lungs or in combination with LRP5 overexpression for 7 days (n=7, mean ± s.e.m., *, p
Figure Legend Snippet: LRP5 mediates age-dependent decline in vascular and alveolar epithelial morphogenesis in the gel implanted on the mouse lungs. (A) Immunofluorescence micrographs showing CD31-positive blood vessels and AQP5-positive alveolar type-I epithelial cells ( top ), CD31-positive blood vessels and SPB-positive alveolar type-II epithelial cells ( 2nd ), CD31-positive blood vessels and VEGFR2 expression ( 3rd ), and CD31-positive blood vessels and Tie2 expression ( bottom ) in the fibrin gel implanted on the 2M vs. 24M old mouse lungs or in combination with LRP5 overexpression for 7 days (scale bar, 20 μm). ( B) Graphs showing quantification of CD31-positive blood vessel numbers ( left ), area of ECs expressing VEGFR2 ( middle ) and Tie2 ( right ) in the gel implanted on the 2M vs. 24M old mouse lungs or in combination with LRP5 overexpression for 7 days (n=7, mean ± s.e.m., *, p

Techniques Used: Immunofluorescence, Expressing, Over Expression

29) Product Images from "Filaggrin null mutations are associated with altered circulating Tregs in atopic dermatitis, et al. Filaggrin null mutations are associated with altered circulating Tregs in atopic dermatitis"

Article Title: Filaggrin null mutations are associated with altered circulating Tregs in atopic dermatitis, et al. Filaggrin null mutations are associated with altered circulating Tregs in atopic dermatitis

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.14031

FLG null mutations are associated with expansion of thymus‐emigrated Tregs in AD. Freshly collected blood cells were analysed by flow cytometry. Live lymphocytes were gated on CD4. Tregs were designated as CD4 + CD25 + CD127 low/– , thymus‐derived (CD45RA + CD31 +/– ) Tregs, mature naïve (CD45RA + CD31 – ) Tregs, and recently thymus‐emigrated (CD45RA + CD31 + . Percentages of (A) thymus‐derived, (B) mature naïve and (C) recently thymus‐emigrated Tregs. Data were analysed using a Student′s t test or one‐way ANOVA followed by a Tukey post‐hoc test with * P
Figure Legend Snippet: FLG null mutations are associated with expansion of thymus‐emigrated Tregs in AD. Freshly collected blood cells were analysed by flow cytometry. Live lymphocytes were gated on CD4. Tregs were designated as CD4 + CD25 + CD127 low/– , thymus‐derived (CD45RA + CD31 +/– ) Tregs, mature naïve (CD45RA + CD31 – ) Tregs, and recently thymus‐emigrated (CD45RA + CD31 + . Percentages of (A) thymus‐derived, (B) mature naïve and (C) recently thymus‐emigrated Tregs. Data were analysed using a Student′s t test or one‐way ANOVA followed by a Tukey post‐hoc test with * P

Techniques Used: Flow Cytometry, Cytometry, Derivative Assay

FLG null‐mutations limit the expansion of circulating effector and memory Tregs in AD. Freshly collected blood cells were analysed by flow cytometry. Live lymphocytes were gated on CD4. Tregs were designated as CD4 + CD25 + CD127 low/– , effector (CD45RA – CCR4 + , and memory (CD45RA – ICOS +/– CD31 +/– . Percentages of (A) CD4 + cells, (B) Tregs, (C) effector Tregs and (D) memory Tregs. Data were analysed using a Student′s t test or one‐way ANOVA followed by a Tukey post‐hoc test with * P
Figure Legend Snippet: FLG null‐mutations limit the expansion of circulating effector and memory Tregs in AD. Freshly collected blood cells were analysed by flow cytometry. Live lymphocytes were gated on CD4. Tregs were designated as CD4 + CD25 + CD127 low/– , effector (CD45RA – CCR4 + , and memory (CD45RA – ICOS +/– CD31 +/– . Percentages of (A) CD4 + cells, (B) Tregs, (C) effector Tregs and (D) memory Tregs. Data were analysed using a Student′s t test or one‐way ANOVA followed by a Tukey post‐hoc test with * P

Techniques Used: Flow Cytometry, Cytometry

30) Product Images from "Autoantigens targeted in scleroderma patients with vascular disease are enriched in endothelial lineage cells"

Article Title: Autoantigens targeted in scleroderma patients with vascular disease are enriched in endothelial lineage cells

Journal: Arthritis & rheumatology (Hoboken, N.J.)

doi: 10.1002/art.39743

Progenitors expressing CD31, a marker of early blood vessels, have high levels of IFI16 and CENP-A expression
Figure Legend Snippet: Progenitors expressing CD31, a marker of early blood vessels, have high levels of IFI16 and CENP-A expression

Techniques Used: Expressing, Marker

31) Product Images from "EXTRACELLULAR VESICLES GENERATED BY PLACENTAL TISSUES EX VIVO: A TRANSPORT SYSTEM FOR IMMUNE MEDIATORS AND GROWTH FACTORS"

Article Title: EXTRACELLULAR VESICLES GENERATED BY PLACENTAL TISSUES EX VIVO: A TRANSPORT SYSTEM FOR IMMUNE MEDIATORS AND GROWTH FACTORS

Journal: American journal of reproductive immunology (New York, N.Y. : 1989)

doi: 10.1111/aji.12860

Distribution of cytokines between the surface and inner volume of EVs from placental villous tissues Distribution between encapsulated and surface cytokines is shown for placental villous cultures. (a) Total EVs isolated by Exoquick™ (b) anti-PLAP MNP-captured EVs; (c) anti-CD31 MNP-captured EVs; (d) anti-HLA-G MNP-captured EVs. Free and EV-associated cytokines are expressed as percent of total (Mean ± SEM, n=5). Blue bars: surface-associated cytokines, red: EV-encapsulated. Multiplexed bead assay measurements on samples collected at day 4 (cumulative amount for days 1–4 of culture).
Figure Legend Snippet: Distribution of cytokines between the surface and inner volume of EVs from placental villous tissues Distribution between encapsulated and surface cytokines is shown for placental villous cultures. (a) Total EVs isolated by Exoquick™ (b) anti-PLAP MNP-captured EVs; (c) anti-CD31 MNP-captured EVs; (d) anti-HLA-G MNP-captured EVs. Free and EV-associated cytokines are expressed as percent of total (Mean ± SEM, n=5). Blue bars: surface-associated cytokines, red: EV-encapsulated. Multiplexed bead assay measurements on samples collected at day 4 (cumulative amount for days 1–4 of culture).

Techniques Used: Isolation

Distribution of growth factors between the surface and inner volume of EVs from placental villous tissues Distribution between encapsulated and surface growth factors is shown for placental villous cultures. (a) Total EVs isolated by Exoquick™; (b) anti-PLAP MNP-captured EVs; (c) anti-CD31 MNP-captured EVs; (d) anti-HLA-G MNP- captured EVs. Free and EV-associated growth factors are expressed as percent of total (Mean ± SEM, n=5). Blue bars: surface-associated growth factors, red: EV-encapsulated. Multiplexed bead assay measurements on samples collected at day 4 (cumulative amount for days 1–4 of culture).
Figure Legend Snippet: Distribution of growth factors between the surface and inner volume of EVs from placental villous tissues Distribution between encapsulated and surface growth factors is shown for placental villous cultures. (a) Total EVs isolated by Exoquick™; (b) anti-PLAP MNP-captured EVs; (c) anti-CD31 MNP-captured EVs; (d) anti-HLA-G MNP- captured EVs. Free and EV-associated growth factors are expressed as percent of total (Mean ± SEM, n=5). Blue bars: surface-associated growth factors, red: EV-encapsulated. Multiplexed bead assay measurements on samples collected at day 4 (cumulative amount for days 1–4 of culture).

Techniques Used: Isolation

32) Product Images from "GPRC5b Modulates Inflammatory Response in Glomerular Diseases via NF-κB Pathway"

Article Title: GPRC5b Modulates Inflammatory Response in Glomerular Diseases via NF-κB Pathway

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2019010089

GPRC5b is enriched in human and mouse podocytes. (A) In human kidney, Western blotting for Gprc5b detects a 40 kDa protein only in the glomerulus (huGlom) and not in rest of the kidney (huROK). Podocin was used to show the purity of the glomerular fraction and GAPDH as a loading control. (B) Gprc5b transcript shows strong enrichment in the glomerulus when compared with the rest of the kidney as detected by conventional PCR. Nephrin and aquaporin 3 were used as glomerular and rest of kidney fraction markers, respectively, whereas 28S gene was used as a loading control. (C) The PCR for Gprc5b in FACS-isolated mouse podocytes (mPod) and rest of glomerulus (mROG) shows an enrichment in podocytes. Nephrin was used to validate the purity of podocyte fractions and GAPDH as a loading control. (D) Immunofluorescence staining for GPRC5b (green) in human kidney cortex shows strong immunoreactivity in glomeruli and no significant signal in extraglomerular areas. (E–G) Double staining of Gprc5b (green) and podocyte foot process marker nephrin (red) shows nearly complete colocalization (yellow). DAPI (blue) was used as a nucleus marker. (H and I) Double labeling with the mesangial marker PDGFR β (red) or with the endothelial marker CD31 (red) does not show significant overlapping reactivity. (J) Immunoelectron microscopy shows gold label for Gprc5b (arrowheads) on podocyte plasma membrane. Magnifications: ×40 in (D), ×200 in (E–G), ×400 in (H and I). Scale bar, 250 nm in (J).
Figure Legend Snippet: GPRC5b is enriched in human and mouse podocytes. (A) In human kidney, Western blotting for Gprc5b detects a 40 kDa protein only in the glomerulus (huGlom) and not in rest of the kidney (huROK). Podocin was used to show the purity of the glomerular fraction and GAPDH as a loading control. (B) Gprc5b transcript shows strong enrichment in the glomerulus when compared with the rest of the kidney as detected by conventional PCR. Nephrin and aquaporin 3 were used as glomerular and rest of kidney fraction markers, respectively, whereas 28S gene was used as a loading control. (C) The PCR for Gprc5b in FACS-isolated mouse podocytes (mPod) and rest of glomerulus (mROG) shows an enrichment in podocytes. Nephrin was used to validate the purity of podocyte fractions and GAPDH as a loading control. (D) Immunofluorescence staining for GPRC5b (green) in human kidney cortex shows strong immunoreactivity in glomeruli and no significant signal in extraglomerular areas. (E–G) Double staining of Gprc5b (green) and podocyte foot process marker nephrin (red) shows nearly complete colocalization (yellow). DAPI (blue) was used as a nucleus marker. (H and I) Double labeling with the mesangial marker PDGFR β (red) or with the endothelial marker CD31 (red) does not show significant overlapping reactivity. (J) Immunoelectron microscopy shows gold label for Gprc5b (arrowheads) on podocyte plasma membrane. Magnifications: ×40 in (D), ×200 in (E–G), ×400 in (H and I). Scale bar, 250 nm in (J).

Techniques Used: Western Blot, Polymerase Chain Reaction, FACS, Isolation, Immunofluorescence, Staining, Double Staining, Marker, Labeling, Immuno-Electron Microscopy

33) Product Images from "Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans"

Article Title: Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans

Journal: Science immunology

doi: 10.1126/sciimmunol.aav7638

rIL-33 stimulation induces ILC2 motility around blood vessels and airways. IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose), over 1 week and culled 24h after the final dose. Live viable precision cut lung slices (PCLS) of 200μm thickness were obtained and stained for CD31 (Magenta, the lung structure and blood vessels), CD4 (cyan, T cells, orange arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow), and time-lapse video taken (1024μm x 1024μm field of view (FOV), 45 min duration under a 20x objective using an inverted confocal microscope) (A) Static image depicting the location of ILC2 and CD4 + T cells, scale bar 100 μm. (B) Zoomed in section of the blood vessel in figure 2A, scale bar 20 μm. (C) High power images of boxed cells in figure 2B showing differences in pattern of cell movement (oscillatory vs amoeboid movement). ILC2 and CD4 + T cells dynamics were tracked and plotted as (D) individual tracks or (E) tracks commencing from centroid and overlaid. (F) Track speed, (G) track length and (H) track displacement were quantified. Representative images shown in (A-C) are from rIL-33 treated mice, where n = 6 mice per treatment (3 slices per mouse were imaged). For (F-H) in box and whiskers plots, each dot represents an individual cell. Data are representative from 4 experiments where n = 6 mice per treatment. *p
Figure Legend Snippet: rIL-33 stimulation induces ILC2 motility around blood vessels and airways. IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose), over 1 week and culled 24h after the final dose. Live viable precision cut lung slices (PCLS) of 200μm thickness were obtained and stained for CD31 (Magenta, the lung structure and blood vessels), CD4 (cyan, T cells, orange arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow), and time-lapse video taken (1024μm x 1024μm field of view (FOV), 45 min duration under a 20x objective using an inverted confocal microscope) (A) Static image depicting the location of ILC2 and CD4 + T cells, scale bar 100 μm. (B) Zoomed in section of the blood vessel in figure 2A, scale bar 20 μm. (C) High power images of boxed cells in figure 2B showing differences in pattern of cell movement (oscillatory vs amoeboid movement). ILC2 and CD4 + T cells dynamics were tracked and plotted as (D) individual tracks or (E) tracks commencing from centroid and overlaid. (F) Track speed, (G) track length and (H) track displacement were quantified. Representative images shown in (A-C) are from rIL-33 treated mice, where n = 6 mice per treatment (3 slices per mouse were imaged). For (F-H) in box and whiskers plots, each dot represents an individual cell. Data are representative from 4 experiments where n = 6 mice per treatment. *p

Techniques Used: Mouse Assay, Staining, Microscopy

Extracellular matrix proteins, collagen-IV and fibronectin, promote increased ILC2 motility. Human ILC2 lines were seeded on tissue culture plates coated with either 10% FBS, fibronectin, collagen-I, collagen-IV or serum free coating (control) for 24h. Cell movement was imaged via the JuLI imaging system and plotted as (A) individual tracks, (B) track speed dot plots and (C) track speed spider plot. IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose) or PBS (25μl), over 1 week and culled 24h after the final dose PCLS obtained. (D) SHG imaging of PCLS revealing collagen fibres, representative maximum intensity projections, scale bar 50μm. (E) GLCM analysis of SHG imaging. (F) Images of Fibronectin expression and localisation. PCLS stained for CD31 (Magenta, the lung structure and blood vessels), Fibronectin (cyan, yellow arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow) and images of 1024μm x 1024μm field of view (FOV) were taken, scale bar 150μm. For panels A-C, n = 3 donors (in triplicate). Data representative of 3 experiments. For panels D-F n = 6 (in triplicate). *p
Figure Legend Snippet: Extracellular matrix proteins, collagen-IV and fibronectin, promote increased ILC2 motility. Human ILC2 lines were seeded on tissue culture plates coated with either 10% FBS, fibronectin, collagen-I, collagen-IV or serum free coating (control) for 24h. Cell movement was imaged via the JuLI imaging system and plotted as (A) individual tracks, (B) track speed dot plots and (C) track speed spider plot. IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose) or PBS (25μl), over 1 week and culled 24h after the final dose PCLS obtained. (D) SHG imaging of PCLS revealing collagen fibres, representative maximum intensity projections, scale bar 50μm. (E) GLCM analysis of SHG imaging. (F) Images of Fibronectin expression and localisation. PCLS stained for CD31 (Magenta, the lung structure and blood vessels), Fibronectin (cyan, yellow arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow) and images of 1024μm x 1024μm field of view (FOV) were taken, scale bar 150μm. For panels A-C, n = 3 donors (in triplicate). Data representative of 3 experiments. For panels D-F n = 6 (in triplicate). *p

Techniques Used: Imaging, Mouse Assay, Expressing, Staining

The number of ILC2 rapidly increase in the peribronchial / perivascular region after rIL-33 treatment. IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose), Alt (10μg) or PBS (25μl) over 1 week and culled 24h after the final dose. The frequency of ILC2 (GFP + CD45 + Lin neg CD3 - NKp46 - CD127 + CD90.2 + KLRG1 + CD25 var IL-13 + IL-5 + ) in the (A) airways (BAL fluid), (B) lung and (C) lung draining lymph nodes (mediastinal). Live viable precision cut lung slices of 200μm thickness were obtained and stained for CD31 (Magenta, the lung structure and blood vessels), CD4 (cyan, T cells, orange arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow). Images of 1024μm x 1024μm field of view (FOV) were taken under a 20x objective using an inverted confocal microscope. (D) Images showing ILC2 (GFP + CD4 - ) CD4+ T cells (CD4+GFP-) location in PBS, rIL-33 and Alt treated mice, scale bar, 150 μm. (E) Number of ILC2 (GFP + CD4 - ) in lung sections per FOV taken under a 10x objective. (F) Schematic illustration of the lung depicting the anatomical location in the lung where precision cut lung slices were prepared. Representative images show two regions of the lung slice from a rIL-33 treated mouse showing distribution of ILC2 and CD4+ T cells, scale bar 150 μm. n = 4 mice per group (Mock(PBS)), n= 6 mice per group (Alt or rIL-33 treatment). Data representative of 4 experiments. *p
Figure Legend Snippet: The number of ILC2 rapidly increase in the peribronchial / perivascular region after rIL-33 treatment. IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose), Alt (10μg) or PBS (25μl) over 1 week and culled 24h after the final dose. The frequency of ILC2 (GFP + CD45 + Lin neg CD3 - NKp46 - CD127 + CD90.2 + KLRG1 + CD25 var IL-13 + IL-5 + ) in the (A) airways (BAL fluid), (B) lung and (C) lung draining lymph nodes (mediastinal). Live viable precision cut lung slices of 200μm thickness were obtained and stained for CD31 (Magenta, the lung structure and blood vessels), CD4 (cyan, T cells, orange arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow). Images of 1024μm x 1024μm field of view (FOV) were taken under a 20x objective using an inverted confocal microscope. (D) Images showing ILC2 (GFP + CD4 - ) CD4+ T cells (CD4+GFP-) location in PBS, rIL-33 and Alt treated mice, scale bar, 150 μm. (E) Number of ILC2 (GFP + CD4 - ) in lung sections per FOV taken under a 10x objective. (F) Schematic illustration of the lung depicting the anatomical location in the lung where precision cut lung slices were prepared. Representative images show two regions of the lung slice from a rIL-33 treated mouse showing distribution of ILC2 and CD4+ T cells, scale bar 150 μm. n = 4 mice per group (Mock(PBS)), n= 6 mice per group (Alt or rIL-33 treatment). Data representative of 4 experiments. *p

Techniques Used: Mouse Assay, Staining, Microscopy

ILC2 utilize distinct chemotactic pathways to home to inflammatory sites in the lung. IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose) or PBS (25μl), over 1 week and culled 24h after the final dose. (A) The percentage of murine ILC2 (CD45 + Lin neg NKp46 - CD3 - ) expressing CCR1, CCR4 and CCR8. CCL8 levels in murine (B) BAL and (C) lung. (D) Location of CCL8 expression and ILC2 and (E) quantified CCL8 deposits in PCLS stained for CD31 (Magenta, the lung structure and blood vessels), CCL8 (cyan, yellow arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow), images of 1024μm x 1024μm FOV, scale bar 150 μm. Human ILC2 lines were generated and migration to varying concentrations of (F) PGD 2 and (G) CCL8 were determined. (H) Peak migratory responses of a human ILC2 cell line to IL-25, TGF-β, rIL-33, CCL8 and PGD 2 . IL13-eGFP mice treated with rIL-33 were also treated with 5μg purified anti-mouse CCR8 antibody i.p., rCCL8 i.n. or an isotype control and PCLS obtained and stained. (I) Localisation of ILC2 in live PCLS. (J) Number of ILC2 per FOV under 10x objective. Time-lapse imaging of 45 min duration was performed and ILC2 (K) track from centroid, (L) track length and (M) track speed and (N) track displacement were quantified. In box and whiskers graphs each data point represents an individual cell. Balb/c mice treated with rIL-33 were further treated with rCCL8, αCCR8 or Isotype control antibody. ( O) Percentage of IL-13 + IL-5 + ILC2 (CD45 + lin - NKp46 - CD3 - GATA-3 + ). ( P) Representation Histogram of MFI of IL-13 and IL-5 and quantification of MFI for (Q) IL-13 and ( R) IL-5 from GATA+ ILC2. For panels A-E n ≥ 4 mice per group. Data representative of 4 experiments. For panels F-H n = 3 individual donors. Data representative of 3 experiments. For panels I-R , n = 5 mice per group. Data representative of 2 experiments *p
Figure Legend Snippet: ILC2 utilize distinct chemotactic pathways to home to inflammatory sites in the lung. IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose) or PBS (25μl), over 1 week and culled 24h after the final dose. (A) The percentage of murine ILC2 (CD45 + Lin neg NKp46 - CD3 - ) expressing CCR1, CCR4 and CCR8. CCL8 levels in murine (B) BAL and (C) lung. (D) Location of CCL8 expression and ILC2 and (E) quantified CCL8 deposits in PCLS stained for CD31 (Magenta, the lung structure and blood vessels), CCL8 (cyan, yellow arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow), images of 1024μm x 1024μm FOV, scale bar 150 μm. Human ILC2 lines were generated and migration to varying concentrations of (F) PGD 2 and (G) CCL8 were determined. (H) Peak migratory responses of a human ILC2 cell line to IL-25, TGF-β, rIL-33, CCL8 and PGD 2 . IL13-eGFP mice treated with rIL-33 were also treated with 5μg purified anti-mouse CCR8 antibody i.p., rCCL8 i.n. or an isotype control and PCLS obtained and stained. (I) Localisation of ILC2 in live PCLS. (J) Number of ILC2 per FOV under 10x objective. Time-lapse imaging of 45 min duration was performed and ILC2 (K) track from centroid, (L) track length and (M) track speed and (N) track displacement were quantified. In box and whiskers graphs each data point represents an individual cell. Balb/c mice treated with rIL-33 were further treated with rCCL8, αCCR8 or Isotype control antibody. ( O) Percentage of IL-13 + IL-5 + ILC2 (CD45 + lin - NKp46 - CD3 - GATA-3 + ). ( P) Representation Histogram of MFI of IL-13 and IL-5 and quantification of MFI for (Q) IL-13 and ( R) IL-5 from GATA+ ILC2. For panels A-E n ≥ 4 mice per group. Data representative of 4 experiments. For panels F-H n = 3 individual donors. Data representative of 3 experiments. For panels I-R , n = 5 mice per group. Data representative of 2 experiments *p

Techniques Used: Mouse Assay, Expressing, Staining, Generated, Migration, Purification, Imaging

34) Product Images from "Pulmonary fibrosis requires cell-autonomous mesenchymal fibroblast growth factor (FGF) signaling"

Article Title: Pulmonary fibrosis requires cell-autonomous mesenchymal fibroblast growth factor (FGF) signaling

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M117.791764

Decreased proliferation and enrichment of Col1α2+ lineage in fibrotic areas in bleomycin-treated Col1a2-CreER; TCKO mice. Col1 α 2-CreER; TCKO; ROSA26 mTmG /+ and control Col1 α 2-CreER; ROSA26 mTmG /+ mice were administered a single dose of intratracheal bleomycin (1.2 units/kg) and lungs were collected 21 days post-bleomycin. Immunohistochemistry for GFP was performed ( A and B ) and the percentage of total fibrotic areas that were GFP+ was analyzed using ImageJ ( C ). Whole lungs were dissociated and the percentage of total live single cells that were GFP+ following bleomycin treatment were analyzed by flow cytometry ( D ). Proliferation was measured by injecting mice with EdU (50 mg/kg i.p.) prior to collection, and dissociated lungs were stained for CD45, CD31, and EpCam (Lin), fixed, permeabilized, and then stained for EdU incorporation. Stained cells were measured by flow cytometry, and EdU incorporation within all single cells ( D ) or Lin-negative cells ( E ) was analyzed. GFP+ cells from bleomycin-treated Col1 α 2-CreER; TCKO; ROSA26 mTmG /+ and control Col1 α 2-CreER; ROSA26 mTmG /+ mice were sorted into 96-well plates (2,000 cells/well) and cultured for 48 h, 5 days, 7 days, and 9 days prior to measurement of total cellular DNA using a CyQuant assay ( F ). * indicates p
Figure Legend Snippet: Decreased proliferation and enrichment of Col1α2+ lineage in fibrotic areas in bleomycin-treated Col1a2-CreER; TCKO mice. Col1 α 2-CreER; TCKO; ROSA26 mTmG /+ and control Col1 α 2-CreER; ROSA26 mTmG /+ mice were administered a single dose of intratracheal bleomycin (1.2 units/kg) and lungs were collected 21 days post-bleomycin. Immunohistochemistry for GFP was performed ( A and B ) and the percentage of total fibrotic areas that were GFP+ was analyzed using ImageJ ( C ). Whole lungs were dissociated and the percentage of total live single cells that were GFP+ following bleomycin treatment were analyzed by flow cytometry ( D ). Proliferation was measured by injecting mice with EdU (50 mg/kg i.p.) prior to collection, and dissociated lungs were stained for CD45, CD31, and EpCam (Lin), fixed, permeabilized, and then stained for EdU incorporation. Stained cells were measured by flow cytometry, and EdU incorporation within all single cells ( D ) or Lin-negative cells ( E ) was analyzed. GFP+ cells from bleomycin-treated Col1 α 2-CreER; TCKO; ROSA26 mTmG /+ and control Col1 α 2-CreER; ROSA26 mTmG /+ mice were sorted into 96-well plates (2,000 cells/well) and cultured for 48 h, 5 days, 7 days, and 9 days prior to measurement of total cellular DNA using a CyQuant assay ( F ). * indicates p

Techniques Used: Mouse Assay, Immunohistochemistry, Flow Cytometry, Cytometry, Staining, Cell Culture, CyQUANT Assay

Isolation and gene expression of Col1α2+ mesenchymal cells. Tamoxifen-treated Col1 α 2-CreER; ROSA26 mTmG /+ mouse lungs were harvested, enzymatically dissociated, and GFP+ cells were identified via flow cytometry. Single cells were identified using forward and side scatter ( A and B ), and GFP+ cells were identified ( D ). Analysis of Cre-negative ROSA26 mTmG /+ mouse lungs was used to confirm gating ( C ). Dissociated lungs were stained with BV421-conjugated antibodies against CD31, CD45, and EpCAM (Lin) ( E ), and abundance of GFP+ cells was measured in both Lin-positive ( F ) and Lin-negative ( G ) populations. PE , phycoerythrin. Total RNA was purified from freshly sorted GFP+ cells and analyzed via quantitative RT-PCR for expression of Fgfr1, Col1 α 1, Col1 α 2, Acta2, Periostin, Pdgfr α, Fgfr2, Fgfr3, and Fgfr4 ( H and I ). GFP+ cells were cultured, and RNA was collected from passage number 2 cells for qRT-PCR analysis of Fgfr1 , Fgfr2 , Fgfr3 , and Fgfr4 ( J ). qRT-PCR data were normalized to Gapdh and expressed as Δ C t . * indicates p
Figure Legend Snippet: Isolation and gene expression of Col1α2+ mesenchymal cells. Tamoxifen-treated Col1 α 2-CreER; ROSA26 mTmG /+ mouse lungs were harvested, enzymatically dissociated, and GFP+ cells were identified via flow cytometry. Single cells were identified using forward and side scatter ( A and B ), and GFP+ cells were identified ( D ). Analysis of Cre-negative ROSA26 mTmG /+ mouse lungs was used to confirm gating ( C ). Dissociated lungs were stained with BV421-conjugated antibodies against CD31, CD45, and EpCAM (Lin) ( E ), and abundance of GFP+ cells was measured in both Lin-positive ( F ) and Lin-negative ( G ) populations. PE , phycoerythrin. Total RNA was purified from freshly sorted GFP+ cells and analyzed via quantitative RT-PCR for expression of Fgfr1, Col1 α 1, Col1 α 2, Acta2, Periostin, Pdgfr α, Fgfr2, Fgfr3, and Fgfr4 ( H and I ). GFP+ cells were cultured, and RNA was collected from passage number 2 cells for qRT-PCR analysis of Fgfr1 , Fgfr2 , Fgfr3 , and Fgfr4 ( J ). qRT-PCR data were normalized to Gapdh and expressed as Δ C t . * indicates p

Techniques Used: Isolation, Expressing, Flow Cytometry, Cytometry, Staining, Purification, Quantitative RT-PCR, Cell Culture

Altered αSMA expression in Col1a2-CreER; TCKO mice following bleomycin treatment. Col1 α 2-CreER; TCKO; ROSA26 mTmG /+ and control Col1 α 2-CreER; ROSA26 mTmG /+ mice were administered a single dose of intratracheal bleomycin (1.2 units/kg) or PBS. ( A–H ) Sections from lungs collected 21 days post-bleomycin were stained for αSMA and GFP. A and E, confocal images were obtained at ×20 magnification for GFP ( green ), αSMA ( red ), and DAPI ( blue ). Higher magnifications of areas indicated by dotted lines are shown for control ( B–D ) and TCKO ( F–H ) mice. Arrows indicate αSMA+ cells. I and J, lungs were dissociated, fixed, permeabilized, stained with BV421-conjugated antibodies against CD45, CD31, and EpAM (Lin) as well as A647-conjugated anti-αSMA antibodies, and analyzed by flow cytometry. Lin-negative cells were analyzed for expression of GFP and αSMA, and the percentage of Lin-negative cells that are αSMA+, GFP- ( I ) and αSMA+, GFP+ ( J ) are shown. * indicates p
Figure Legend Snippet: Altered αSMA expression in Col1a2-CreER; TCKO mice following bleomycin treatment. Col1 α 2-CreER; TCKO; ROSA26 mTmG /+ and control Col1 α 2-CreER; ROSA26 mTmG /+ mice were administered a single dose of intratracheal bleomycin (1.2 units/kg) or PBS. ( A–H ) Sections from lungs collected 21 days post-bleomycin were stained for αSMA and GFP. A and E, confocal images were obtained at ×20 magnification for GFP ( green ), αSMA ( red ), and DAPI ( blue ). Higher magnifications of areas indicated by dotted lines are shown for control ( B–D ) and TCKO ( F–H ) mice. Arrows indicate αSMA+ cells. I and J, lungs were dissociated, fixed, permeabilized, stained with BV421-conjugated antibodies against CD45, CD31, and EpAM (Lin) as well as A647-conjugated anti-αSMA antibodies, and analyzed by flow cytometry. Lin-negative cells were analyzed for expression of GFP and αSMA, and the percentage of Lin-negative cells that are αSMA+, GFP- ( I ) and αSMA+, GFP+ ( J ) are shown. * indicates p

Techniques Used: Expressing, Mouse Assay, Staining, Flow Cytometry, Cytometry

35) Product Images from "CD47 prevents the elimination of diseased fibroblasts in scleroderma"

Article Title: CD47 prevents the elimination of diseased fibroblasts in scleroderma

Journal: bioRxiv

doi: 10.1101/2020.06.06.138222

JUN expands distinct fibroblast populations in a hedgehog dependent manner. (A) Representative trichrome stains and immunofluorescence stains against pJUN and FSP1 without JUN induction (-JUN), with JUN induction (+JUN) and after bleomycin injection. Black scale bar = 500 µm. White scale bar = 75 µm. (B) Corresponding quantification of dermal thickness, fat layer thickness, fat/skin thickness relationship, fibrotic areas, total CD31+ endothelial cells and dermal pJUN+ cells/Field of view. Turkey’s multiple comparisons test. * p
Figure Legend Snippet: JUN expands distinct fibroblast populations in a hedgehog dependent manner. (A) Representative trichrome stains and immunofluorescence stains against pJUN and FSP1 without JUN induction (-JUN), with JUN induction (+JUN) and after bleomycin injection. Black scale bar = 500 µm. White scale bar = 75 µm. (B) Corresponding quantification of dermal thickness, fat layer thickness, fat/skin thickness relationship, fibrotic areas, total CD31+ endothelial cells and dermal pJUN+ cells/Field of view. Turkey’s multiple comparisons test. * p

Techniques Used: Immunofluorescence, Injection

36) Product Images from "CXCR4-binding PET tracers link monocyte recruitment and endothelial injury in murine atherosclerosis"

Article Title: CXCR4-binding PET tracers link monocyte recruitment and endothelial injury in murine atherosclerosis

Journal: bioRxiv

doi: 10.1101/2020.01.02.892935

CXCR4 accounts substantially for the PET signal arising from the 64 Cu-DOTA-vMIP-II tracer. (A) CXCR4 expression in aortic sinus from Apoe -/- mice on HFD. Scale bar: 500 μm (upper) and 50 μm (bottom) (B) CXCR4 distribution in whole thoracic aorta from Apoe -/- mice on HFD. Scale bar: 1 mm (C) CXCR4 distribution in the plaque at the aortic arch from Apoe -/- mice on HFD. Scale bar: 200 μm (D) Representative images of CXCR4 expression in aortic sinus from Apoe -/- mice on HFD after 2 weeks of PBS (upper) or AAV-mApoE (bottom) treatment. Scale bar: 500 μm, and (E) quantification of CXCR4 + CD31 + areas (n=6 per group). (F) Representative histogram of CXCR4 expression on aortic ECs from Apoe -/- mice on HFD after 2 weeks of PBS (red) or AAV-mApoE (blue) treatment. Yellow histogram is isotype control. (G) The percentage of CXCR4 hi ECs in total aortic ECs with or without 2 weeks of AAV-mApoE treatment (n=7 per group). (H) Representative 64 Cu-DOTA-vMIP-II PET images and (I) quantification (n=5 per group) at aortic arches of Apoe -/- mice on HFD with or without CXCR4 blocking by 0.5 mg of FC131 (n=5 per group). All data are shown as means ± SEM. *p
Figure Legend Snippet: CXCR4 accounts substantially for the PET signal arising from the 64 Cu-DOTA-vMIP-II tracer. (A) CXCR4 expression in aortic sinus from Apoe -/- mice on HFD. Scale bar: 500 μm (upper) and 50 μm (bottom) (B) CXCR4 distribution in whole thoracic aorta from Apoe -/- mice on HFD. Scale bar: 1 mm (C) CXCR4 distribution in the plaque at the aortic arch from Apoe -/- mice on HFD. Scale bar: 200 μm (D) Representative images of CXCR4 expression in aortic sinus from Apoe -/- mice on HFD after 2 weeks of PBS (upper) or AAV-mApoE (bottom) treatment. Scale bar: 500 μm, and (E) quantification of CXCR4 + CD31 + areas (n=6 per group). (F) Representative histogram of CXCR4 expression on aortic ECs from Apoe -/- mice on HFD after 2 weeks of PBS (red) or AAV-mApoE (blue) treatment. Yellow histogram is isotype control. (G) The percentage of CXCR4 hi ECs in total aortic ECs with or without 2 weeks of AAV-mApoE treatment (n=7 per group). (H) Representative 64 Cu-DOTA-vMIP-II PET images and (I) quantification (n=5 per group) at aortic arches of Apoe -/- mice on HFD with or without CXCR4 blocking by 0.5 mg of FC131 (n=5 per group). All data are shown as means ± SEM. *p

Techniques Used: Positron Emission Tomography, Expressing, Mouse Assay, Blocking Assay

37) Product Images from "Loss of Renal Peritubular Capillaries in Hypertensive Patients is Detectable by Urinary Endothelial Microparticle Levels"

Article Title: Loss of Renal Peritubular Capillaries in Hypertensive Patients is Detectable by Urinary Endothelial Microparticle Levels

Journal: Hypertension (Dallas, Tex. : 1979)

doi: 10.1161/HYPERTENSIONAHA.118.11766

Levels of exosomes in urine of hypertensive patients. (A) There were no differences among the groups in percent of urinary PL-VAP + /CD31 + , PL-VAP + /CD144 + , and PL-VAP + /CD31 + /CD144 + exosomes. (B) PTC-EMPs were identified using flow cytometry as PL-VAP + /CD31 − /CD144 − as shown in representative fluorescent images. Scale bar =20 µm. (C) Renal vein and systemic levels of PL-VAP + /CD31 − /CD144 − EMPs were not different among the groups, whereas their urinary levels were elevated in both EH and RVH compared to HVs (p
Figure Legend Snippet: Levels of exosomes in urine of hypertensive patients. (A) There were no differences among the groups in percent of urinary PL-VAP + /CD31 + , PL-VAP + /CD144 + , and PL-VAP + /CD31 + /CD144 + exosomes. (B) PTC-EMPs were identified using flow cytometry as PL-VAP + /CD31 − /CD144 − as shown in representative fluorescent images. Scale bar =20 µm. (C) Renal vein and systemic levels of PL-VAP + /CD31 − /CD144 − EMPs were not different among the groups, whereas their urinary levels were elevated in both EH and RVH compared to HVs (p

Techniques Used: Flow Cytometry, Cytometry

38) Product Images from "LKB1 inactivation modulates chromatin accessibility to drive metastatic progression"

Article Title: LKB1 inactivation modulates chromatin accessibility to drive metastatic progression

Journal: bioRxiv

doi: 10.1101/2021.03.29.437560

Genotype-specific activation of SOX17 expression in metastatic, LKB1-deficient cells. a. Schematic of tumor initiation, sample processing, and multi-omic profiling. Lentiviral Cre initiates tumors in Kras LSL-G12D ;p53 flox/flox ;Rosa26 LSL-tdTomato ( KPT ) mice with and without homozygous Lkb1 flox/flox alleles. tdTomato+ cancer cells negative for the lineage markers CD45, CD31, F4/F80, and Ter119 were sorted by FACS before library preparation for ATAC-seq, scATAC-seq, and RNA-seq. b. PCA of the top 10,000 variable ATAC-seq peaks across 25 primary tumor samples. Technical replicates are averaged. c. Comparison of the changes in motif accessibility (ΔchromVAR Deviation Scores) across LKB1-proficient(x-axis) and LKB1-deficient (y-axis) metastases compared to primary tumors of the same genotype. Dark grey or colored points are called significantly different (q
Figure Legend Snippet: Genotype-specific activation of SOX17 expression in metastatic, LKB1-deficient cells. a. Schematic of tumor initiation, sample processing, and multi-omic profiling. Lentiviral Cre initiates tumors in Kras LSL-G12D ;p53 flox/flox ;Rosa26 LSL-tdTomato ( KPT ) mice with and without homozygous Lkb1 flox/flox alleles. tdTomato+ cancer cells negative for the lineage markers CD45, CD31, F4/F80, and Ter119 were sorted by FACS before library preparation for ATAC-seq, scATAC-seq, and RNA-seq. b. PCA of the top 10,000 variable ATAC-seq peaks across 25 primary tumor samples. Technical replicates are averaged. c. Comparison of the changes in motif accessibility (ΔchromVAR Deviation Scores) across LKB1-proficient(x-axis) and LKB1-deficient (y-axis) metastases compared to primary tumors of the same genotype. Dark grey or colored points are called significantly different (q

Techniques Used: Activation Assay, Expressing, Mouse Assay, FACS, RNA Sequencing Assay

LKB1-deficient primary tumors harbor sub-populations of SOX17+ cells. a. Representative immunohistochemistry for SOX17 (in brown) and tdTomato (in grey) on two LKB1-deficient lung adenocarcinoma primary tumors. Scale bars represent 50uM. b. Schematic of tumor initiation and processing for scATAC-seq. tdTomato+, DAPI-cancer cells that were negative for the lineage (Lin) markers CD45, CD31, F4/F80, and Ter119 were sorted by FACS before scATAC-seq library preparation. c. Uniform Manifold Approximation and Projection (UMAP) of 8392 nuclei from 4 KPT primary tumors and 6021 nuclei from 3 KPT;Lkb1 −/− primary tumors, colored by genotype ( left ) or cluster according to Seurat graph clustering ( right ). d. Nkx2.1 and Sox17 genome accessibility tracks for each cluster indicated in Fig 4c . Significant ATAC-seq peaks from bulk chromatin accessibility profiling ( Fig. 3e ) are highlighted in grey and indicated with an asterisk (*). e and f. UMAP colored by the average gene body accessibility for Nkx2.1 ( e ) or Sox17 ( f ) in each cell. g and h. Top : Footprint of accessibility for each scATAC-seq cluster for genomic regions containing NKX2 ( g ) and SOX ( h ) motifs. Bottom : Modeled hexamer insertion bias of Tn5 around sites containing each motif.
Figure Legend Snippet: LKB1-deficient primary tumors harbor sub-populations of SOX17+ cells. a. Representative immunohistochemistry for SOX17 (in brown) and tdTomato (in grey) on two LKB1-deficient lung adenocarcinoma primary tumors. Scale bars represent 50uM. b. Schematic of tumor initiation and processing for scATAC-seq. tdTomato+, DAPI-cancer cells that were negative for the lineage (Lin) markers CD45, CD31, F4/F80, and Ter119 were sorted by FACS before scATAC-seq library preparation. c. Uniform Manifold Approximation and Projection (UMAP) of 8392 nuclei from 4 KPT primary tumors and 6021 nuclei from 3 KPT;Lkb1 −/− primary tumors, colored by genotype ( left ) or cluster according to Seurat graph clustering ( right ). d. Nkx2.1 and Sox17 genome accessibility tracks for each cluster indicated in Fig 4c . Significant ATAC-seq peaks from bulk chromatin accessibility profiling ( Fig. 3e ) are highlighted in grey and indicated with an asterisk (*). e and f. UMAP colored by the average gene body accessibility for Nkx2.1 ( e ) or Sox17 ( f ) in each cell. g and h. Top : Footprint of accessibility for each scATAC-seq cluster for genomic regions containing NKX2 ( g ) and SOX ( h ) motifs. Bottom : Modeled hexamer insertion bias of Tn5 around sites containing each motif.

Techniques Used: Immunohistochemistry, FACS

39) Product Images from "A microfluidic platform for quantitative analysis of cancer angiogenesis and intravasation a)"

Article Title: A microfluidic platform for quantitative analysis of cancer angiogenesis and intravasation a)

Journal: Biomicrofluidics

doi: 10.1063/1.4894595

Micrographs and quantification of the cancer intravasation assay. (a) Micrographs of a cancer cell trans-migrating through the microvessel wall. (Red: CD31, Green: MDA-MB-231, and Blue: Nuclei) Fluorescence and DIC micrographs show that the cancer cell
Figure Legend Snippet: Micrographs and quantification of the cancer intravasation assay. (a) Micrographs of a cancer cell trans-migrating through the microvessel wall. (Red: CD31, Green: MDA-MB-231, and Blue: Nuclei) Fluorescence and DIC micrographs show that the cancer cell

Techniques Used: Multiple Displacement Amplification, Fluorescence

40) Product Images from "FGFR2 Is Required for AEC2 Homeostasis and Survival after Bleomycin-induced Lung Injury"

Article Title: FGFR2 Is Required for AEC2 Homeostasis and Survival after Bleomycin-induced Lung Injury

Journal: American Journal of Respiratory Cell and Molecular Biology

doi: 10.1165/rcmb.2019-0079OC

AEC2-specific deletion of FGFRs results in a significant loss of lineage-labeled AECs after bleomycin. SPC-TCKO and control mice were administered an injection with tamoxifen at 6 weeks of age, and at 8 weeks of age, they were treated with a single dose of intratracheal bleomycin (0.5 U/kg). Lungs from mice 7 days after bleomycin administration were dissociated; immunostained for CD45, CD31, and EpCAM (epithelial cell adhesion molecule); and analyzed via flow cytometry. ( A–C ) GFP + cells as a percentage of total CD45 − ,CD31 − single cells ( A ), or CD45 − ,CD31 − ,EpCAM + single cells ( B ), and EpCAM + cells as a percentage of total CD45 − ,CD31 − cells ( C ) are shown. Lungs from mice 7 days after bleomycin administration were inflation fixed with 4% paraformaldehyde, cryopreserved, and embedded in optimal cutting temperature compound for frozen sections. ( D ) Confocal imaging of frozen sections immunostained for GFP, pro-SPC, PDPN (podoplanin), and counterstained with DAPI. Scale bars: 200 μm. ( E – G ) Quantification of GFP + ,PDPN + and GFP + ,SPC + ,PDPN + cells was performed and analyzed using ImageJ software. Bars represent ± SEM. * P
Figure Legend Snippet: AEC2-specific deletion of FGFRs results in a significant loss of lineage-labeled AECs after bleomycin. SPC-TCKO and control mice were administered an injection with tamoxifen at 6 weeks of age, and at 8 weeks of age, they were treated with a single dose of intratracheal bleomycin (0.5 U/kg). Lungs from mice 7 days after bleomycin administration were dissociated; immunostained for CD45, CD31, and EpCAM (epithelial cell adhesion molecule); and analyzed via flow cytometry. ( A–C ) GFP + cells as a percentage of total CD45 − ,CD31 − single cells ( A ), or CD45 − ,CD31 − ,EpCAM + single cells ( B ), and EpCAM + cells as a percentage of total CD45 − ,CD31 − cells ( C ) are shown. Lungs from mice 7 days after bleomycin administration were inflation fixed with 4% paraformaldehyde, cryopreserved, and embedded in optimal cutting temperature compound for frozen sections. ( D ) Confocal imaging of frozen sections immunostained for GFP, pro-SPC, PDPN (podoplanin), and counterstained with DAPI. Scale bars: 200 μm. ( E – G ) Quantification of GFP + ,PDPN + and GFP + ,SPC + ,PDPN + cells was performed and analyzed using ImageJ software. Bars represent ± SEM. * P

Techniques Used: Labeling, Mouse Assay, Injection, Flow Cytometry, Imaging, Software

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    BioLegend cd3
    iαβTs expand in PDA. (a) CD45 + leukocytes infiltrating day 21 orthotopic KPC tumors, normal pancreas, and spleens in WT mice were gated and tested for the frequency of TCRβ + CD4 – CD8 – NK1.1 – iαβTs. Representative contour plots and quantitative data are shown (n=10). (b) CD45 + TCRβ + NK1.1 – leukocytes from pancreata and spleens of 6 month-old KC mice were gated and tested for co-expression CD4 and CD8. Representative contour plots are shown (n=5). (c) Multiplex IHC of human PDA and adjacent normal pancreas were stained for CK19, <t>CD3,</t> CD4, and CD8. The frequency of CD3 + CD4 – CD8 – cells were quantified and representative images are shown. (d) Orthotopic KPC tumors were harvested from WT mice on day 21. CD45 + CD3 + leukocytes were purified by FACS and analyzed by single cell RNAseq. The distribution of cellular clusters was determined using the t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm. Each cluster is identified by a distinct color. Percent cellular abundance in each cluster is indicated. (e) Orthotopic KPC tumors were harvested from WT mice on days 7, 14, or 21 after tumor cell implantation and tumor-infiltrating CD45 + TCRβ + CD4 – CD8 – leukocytes were gated and tested for expression of NK1.1. Representative contour plots from days 7 and 21 and quantitative data comparing frequency of tumor-infiltrating iαβT per NKT cells at all time points are shown (n=5/time point). (f) Paraffin-embedded sections made from tumors of mice serially treated with anti-TCRγ/δ and NK1.1 depleting antibodies were tested for co-expression of Hematoxylin, CD3, CD4, and CD8 in the PDA TME. (g) CD45 + TCRβ + NK1.1 – leukocytes infiltrating orthotopic KPC tumors in WT and Fas lpr mice were gated and tested for expression of CD4 and CD8. Representative contour plots and quantitative data are shown (n=5/group). (h) The thymus from 6-month old WT and KC mice were harvested and CD45 + TCRβ + thymocytes were gated and tested for expression of CD4 and CD8. The frequency of iαβTs in the thymus was calculated (n=5/group). (i) CD4 + T cells, CD8 + T cells, or iαβTs were harvested from CD45.1 mice and transferred i.v. to orthotopic PDA-bearing CD45.2 mice. PDA tumors were harvested at 96 hours and CD45.1 + cells were gated and tested for CD4 and CD8 expression. Representative contour plots are shown (n=5/group). (j) WT mice were orthotopically administered KPC tumor cells and sacrificed on day 21. PDA-infiltrating CD4 + T cells, CD8 + T cells, and iαβTs were assayed for Ki67 proliferative index. Representative contour plots and quantitative data are shown (n=5). (k) Splenic and orthotopic PDA-infiltrating iαβTs were tested on day 21 for expression of CCR2, CCR5, and CCR6 (n=5). (l) The frequency of PDA-infiltrating iαβTs was tested on day 21 in WT, CCR2 –/– , CCR5 –/– , and CCR6 –/– hosts (n=5/group). All experiments were repeated at least 3 times (*p
    Cd3, supplied by BioLegend, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend cd31
    Levels of exosomes in urine of hypertensive patients. (A) There were no differences among the groups in percent of urinary PL-VAP + <t>/CD31</t> + , PL-VAP + /CD144 + , and PL-VAP + /CD31 + /CD144 + exosomes. (B) PTC-EMPs were identified using flow cytometry as PL-VAP + /CD31 − /CD144 − as shown in representative fluorescent images. Scale bar =20 µm. (C) Renal vein and systemic levels of PL-VAP + /CD31 − /CD144 − EMPs were not different among the groups, whereas their urinary levels were elevated in both EH and RVH compared to HVs (p
    Cd31, supplied by BioLegend, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend biotin anti mouse cd31
    Examples of histologically stained tumor sections. The upper row shows NADH-diaphorase stained tumor sections of an untreated tumor, a fully treated tumor, and a partially treated tumor. Treated tumors were excised at 24 h after PDT. The approximate angle of light incidence during PDT is indicated by the arrows. For the partially treated tumor, the black cross roughly indicates the part of the light beam that was blocked by black paper covering the skin. The middle row shows magnifications of H E, NADH-diaphorase, and <t>CD31</t> stained sections of an untreated tumor. The fluorescence signal of the CD31 detection is shown in red, while the Hoechst 33342 dye injected 5 min before animal sacrifice is shown in blue. The bottom row shows the same types of images for a treated tumor.
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    BioLegend rat anti mouse cd31
    The pY747 peptide inhibits VEGFR2-induced angiogenesis in ex vivo and in vivo . a ) Inhibition of ex vivo endothelial sprouting by the pY747 peptide. Mouse aortic rings were embedded in matrigel in the presence or absence of 40 ng/mL of VEGF and peptides as indicated. Photographs were taken at three days and the number of endothelial sprouts originating from each ring was determined. b ) Quantification of aortic ring assay as indicated in Fig. 3a . c ) Inhibition of in vivo angiogenesis by pY747 peptide. Results of matrigel plug angiogenesis assay are shown. The indicated peptides at 200 µM concentration were mixed with growth factor-reduced matrigel containing VEGF (500 ng/mL) and injected subcutaneously into wild type mice. Seven days later, the matrigel implants were removed, sectioned, and blood vessels were stained with <t>CD31</t> Ab (red) and nuclei with DAPI (blue). Vessel area was determined using ImagePro. d ) Quantified results of matrigel plug assay as indicated in Fig. 3c .
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    iαβTs expand in PDA. (a) CD45 + leukocytes infiltrating day 21 orthotopic KPC tumors, normal pancreas, and spleens in WT mice were gated and tested for the frequency of TCRβ + CD4 – CD8 – NK1.1 – iαβTs. Representative contour plots and quantitative data are shown (n=10). (b) CD45 + TCRβ + NK1.1 – leukocytes from pancreata and spleens of 6 month-old KC mice were gated and tested for co-expression CD4 and CD8. Representative contour plots are shown (n=5). (c) Multiplex IHC of human PDA and adjacent normal pancreas were stained for CK19, CD3, CD4, and CD8. The frequency of CD3 + CD4 – CD8 – cells were quantified and representative images are shown. (d) Orthotopic KPC tumors were harvested from WT mice on day 21. CD45 + CD3 + leukocytes were purified by FACS and analyzed by single cell RNAseq. The distribution of cellular clusters was determined using the t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm. Each cluster is identified by a distinct color. Percent cellular abundance in each cluster is indicated. (e) Orthotopic KPC tumors were harvested from WT mice on days 7, 14, or 21 after tumor cell implantation and tumor-infiltrating CD45 + TCRβ + CD4 – CD8 – leukocytes were gated and tested for expression of NK1.1. Representative contour plots from days 7 and 21 and quantitative data comparing frequency of tumor-infiltrating iαβT per NKT cells at all time points are shown (n=5/time point). (f) Paraffin-embedded sections made from tumors of mice serially treated with anti-TCRγ/δ and NK1.1 depleting antibodies were tested for co-expression of Hematoxylin, CD3, CD4, and CD8 in the PDA TME. (g) CD45 + TCRβ + NK1.1 – leukocytes infiltrating orthotopic KPC tumors in WT and Fas lpr mice were gated and tested for expression of CD4 and CD8. Representative contour plots and quantitative data are shown (n=5/group). (h) The thymus from 6-month old WT and KC mice were harvested and CD45 + TCRβ + thymocytes were gated and tested for expression of CD4 and CD8. The frequency of iαβTs in the thymus was calculated (n=5/group). (i) CD4 + T cells, CD8 + T cells, or iαβTs were harvested from CD45.1 mice and transferred i.v. to orthotopic PDA-bearing CD45.2 mice. PDA tumors were harvested at 96 hours and CD45.1 + cells were gated and tested for CD4 and CD8 expression. Representative contour plots are shown (n=5/group). (j) WT mice were orthotopically administered KPC tumor cells and sacrificed on day 21. PDA-infiltrating CD4 + T cells, CD8 + T cells, and iαβTs were assayed for Ki67 proliferative index. Representative contour plots and quantitative data are shown (n=5). (k) Splenic and orthotopic PDA-infiltrating iαβTs were tested on day 21 for expression of CCR2, CCR5, and CCR6 (n=5). (l) The frequency of PDA-infiltrating iαβTs was tested on day 21 in WT, CCR2 –/– , CCR5 –/– , and CCR6 –/– hosts (n=5/group). All experiments were repeated at least 3 times (*p

    Journal: Cancer discovery

    Article Title: Innate αβ T cells Mediate Anti-tumor Immunity by Orchestrating Immunogenic Macrophage Programming

    doi: 10.1158/2159-8290.CD-19-0161

    Figure Lengend Snippet: iαβTs expand in PDA. (a) CD45 + leukocytes infiltrating day 21 orthotopic KPC tumors, normal pancreas, and spleens in WT mice were gated and tested for the frequency of TCRβ + CD4 – CD8 – NK1.1 – iαβTs. Representative contour plots and quantitative data are shown (n=10). (b) CD45 + TCRβ + NK1.1 – leukocytes from pancreata and spleens of 6 month-old KC mice were gated and tested for co-expression CD4 and CD8. Representative contour plots are shown (n=5). (c) Multiplex IHC of human PDA and adjacent normal pancreas were stained for CK19, CD3, CD4, and CD8. The frequency of CD3 + CD4 – CD8 – cells were quantified and representative images are shown. (d) Orthotopic KPC tumors were harvested from WT mice on day 21. CD45 + CD3 + leukocytes were purified by FACS and analyzed by single cell RNAseq. The distribution of cellular clusters was determined using the t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm. Each cluster is identified by a distinct color. Percent cellular abundance in each cluster is indicated. (e) Orthotopic KPC tumors were harvested from WT mice on days 7, 14, or 21 after tumor cell implantation and tumor-infiltrating CD45 + TCRβ + CD4 – CD8 – leukocytes were gated and tested for expression of NK1.1. Representative contour plots from days 7 and 21 and quantitative data comparing frequency of tumor-infiltrating iαβT per NKT cells at all time points are shown (n=5/time point). (f) Paraffin-embedded sections made from tumors of mice serially treated with anti-TCRγ/δ and NK1.1 depleting antibodies were tested for co-expression of Hematoxylin, CD3, CD4, and CD8 in the PDA TME. (g) CD45 + TCRβ + NK1.1 – leukocytes infiltrating orthotopic KPC tumors in WT and Fas lpr mice were gated and tested for expression of CD4 and CD8. Representative contour plots and quantitative data are shown (n=5/group). (h) The thymus from 6-month old WT and KC mice were harvested and CD45 + TCRβ + thymocytes were gated and tested for expression of CD4 and CD8. The frequency of iαβTs in the thymus was calculated (n=5/group). (i) CD4 + T cells, CD8 + T cells, or iαβTs were harvested from CD45.1 mice and transferred i.v. to orthotopic PDA-bearing CD45.2 mice. PDA tumors were harvested at 96 hours and CD45.1 + cells were gated and tested for CD4 and CD8 expression. Representative contour plots are shown (n=5/group). (j) WT mice were orthotopically administered KPC tumor cells and sacrificed on day 21. PDA-infiltrating CD4 + T cells, CD8 + T cells, and iαβTs were assayed for Ki67 proliferative index. Representative contour plots and quantitative data are shown (n=5). (k) Splenic and orthotopic PDA-infiltrating iαβTs were tested on day 21 for expression of CCR2, CCR5, and CCR6 (n=5). (l) The frequency of PDA-infiltrating iαβTs was tested on day 21 in WT, CCR2 –/– , CCR5 –/– , and CCR6 –/– hosts (n=5/group). All experiments were repeated at least 3 times (*p

    Article Snippet: Fluorescently labelled antibodies for CD45 (2D1), TCRαβ (IP26), CD4 (A161A1), CD8 (HIT1a), HLA-DR (L243), TNFα (Mab11), CD11B (ICR F44), IFN-γ (4S.B3), IL-10 (JES3–9D7), ICOS (C398.4A), CD3 (OKT3), CD44 (IM7; all Biolegend) and CD86 (2331FUN-1; BD Biosciences) we used to stain for analysis.

    Techniques: Mouse Assay, Expressing, Multiplex Assay, Immunohistochemistry, Staining, Purification, FACS

    iαβTs induce T cell dependent tumor immunity but are directly suppressive to conventional T cells. (a) WT mice bearing orthotopic KPC tumors were sacrificed on day 21. Splenic and PDA-infiltrating iαβTs were tested for expression of FasL, Perforin, and Granzyme B (n=5 mice). (b, c) iαβTs were harvested by FACS from orthotopic PDA tumors and cultured in various ratio with KPC tumor cells. (b) Proliferation of KPC tumors cells was tested using the XTT assay. (c) Cytotoxicity against KPC tumor cells was determined in an LDH release assay. (d) iαβTs were harvested by FACS from orthotopic PDA tumors and cultured in 1:1 ratio with KPC tumor cells. KPC tumor cell apoptosis was determined by co-staining for Annexin V and PI. (e) WT mice were orthotopically administered KPC tumor cells admixed with iαβTs. Cohorts were either serially depleted of both CD4 + and CD8 + T cells or administered isotype control before sacrifice on day 21. Representative images and quantitative analysis of tumor weights are shown (n=5/group). This experiment was repeated twice. (f-i) Polyclonal splenic CD4 + or CD8 + T cells were cultured without stimulation, stimulated by CD3/CD28 co-ligation, or stimulated by CD3/CD28 co-ligation in co-culture with iαβTs. CD4 + and CD8 + T cell activation were determined at 72h by their expression of (f) IFNγ, (g) TNFα, (h) T-bet, and (i) CD69. Representative contour plots and quantitative data are shown. (j) Polyclonal splenic CD3 + T cells were stimulated by CD3/CD28 co-ligation, either alone or in co-culture with iαβTs. Cell culture supernatant was tested for expression of IFNγ, TNFα, and IL-2 at 72h. (k) Polyclonal splenic CD4 + T cells were cultured without stimulation, stimulated by CD3/CD28 co-ligation, or stimulated by CD3/CD28 co-ligation in co-culture with iαβTs. CD4 + T cells were tested for expression of FoxP3 at 72h. (l) Spleen and PDA-infiltrating iαβTs were tested for expression of PD-L1 by flow cytometry. (m, n) Polyclonal splenic CD4 + T cells from PD-L1 –/– mice were cultured without stimulation, stimulated by CD3/CD28 co-ligation, or stimulated by CD3/CD28 co-ligation in co-culture with WT iαβTs, either alone or with an αPD-L1 neutralizing mAb. CD4 + T cells were tested for expression of (m) CD44 and (n) IFNγ at 72h. Experiments were performed in replicates of 5 and repeated at least 4 times (*p

    Journal: Cancer discovery

    Article Title: Innate αβ T cells Mediate Anti-tumor Immunity by Orchestrating Immunogenic Macrophage Programming

    doi: 10.1158/2159-8290.CD-19-0161

    Figure Lengend Snippet: iαβTs induce T cell dependent tumor immunity but are directly suppressive to conventional T cells. (a) WT mice bearing orthotopic KPC tumors were sacrificed on day 21. Splenic and PDA-infiltrating iαβTs were tested for expression of FasL, Perforin, and Granzyme B (n=5 mice). (b, c) iαβTs were harvested by FACS from orthotopic PDA tumors and cultured in various ratio with KPC tumor cells. (b) Proliferation of KPC tumors cells was tested using the XTT assay. (c) Cytotoxicity against KPC tumor cells was determined in an LDH release assay. (d) iαβTs were harvested by FACS from orthotopic PDA tumors and cultured in 1:1 ratio with KPC tumor cells. KPC tumor cell apoptosis was determined by co-staining for Annexin V and PI. (e) WT mice were orthotopically administered KPC tumor cells admixed with iαβTs. Cohorts were either serially depleted of both CD4 + and CD8 + T cells or administered isotype control before sacrifice on day 21. Representative images and quantitative analysis of tumor weights are shown (n=5/group). This experiment was repeated twice. (f-i) Polyclonal splenic CD4 + or CD8 + T cells were cultured without stimulation, stimulated by CD3/CD28 co-ligation, or stimulated by CD3/CD28 co-ligation in co-culture with iαβTs. CD4 + and CD8 + T cell activation were determined at 72h by their expression of (f) IFNγ, (g) TNFα, (h) T-bet, and (i) CD69. Representative contour plots and quantitative data are shown. (j) Polyclonal splenic CD3 + T cells were stimulated by CD3/CD28 co-ligation, either alone or in co-culture with iαβTs. Cell culture supernatant was tested for expression of IFNγ, TNFα, and IL-2 at 72h. (k) Polyclonal splenic CD4 + T cells were cultured without stimulation, stimulated by CD3/CD28 co-ligation, or stimulated by CD3/CD28 co-ligation in co-culture with iαβTs. CD4 + T cells were tested for expression of FoxP3 at 72h. (l) Spleen and PDA-infiltrating iαβTs were tested for expression of PD-L1 by flow cytometry. (m, n) Polyclonal splenic CD4 + T cells from PD-L1 –/– mice were cultured without stimulation, stimulated by CD3/CD28 co-ligation, or stimulated by CD3/CD28 co-ligation in co-culture with WT iαβTs, either alone or with an αPD-L1 neutralizing mAb. CD4 + T cells were tested for expression of (m) CD44 and (n) IFNγ at 72h. Experiments were performed in replicates of 5 and repeated at least 4 times (*p

    Article Snippet: Fluorescently labelled antibodies for CD45 (2D1), TCRαβ (IP26), CD4 (A161A1), CD8 (HIT1a), HLA-DR (L243), TNFα (Mab11), CD11B (ICR F44), IFN-γ (4S.B3), IL-10 (JES3–9D7), ICOS (C398.4A), CD3 (OKT3), CD44 (IM7; all Biolegend) and CD86 (2331FUN-1; BD Biosciences) we used to stain for analysis.

    Techniques: Mouse Assay, Expressing, FACS, Cell Culture, XTT Assay, Lactate Dehydrogenase Assay, Staining, Ligation, Co-Culture Assay, Activation Assay, Flow Cytometry

    iαβTs infiltrating PDA are phenotypically distinct. WT mice bearing orthotopic KPC tumors were sacrificed on day 21. (a-g) Splenic and PDA-infiltrating iαβTs were tested for expression of (a) JAML, (b) CD107a, (c) CTLA-4, (d) TIM-3, (e) PD-1, (f) CD39, (g) CD40L, LAG-3, CD73, ICOS, and Dectin-1. (h) Splenic and PDA-infiltrating iαβTs were tested for expression of CD62L. (i ) TCR sequencing of splenic iαβTs and PDA-infiltrating iαβTs, CD4 + and CD8 + T cells was performed in triplicate and assessed for overlapping clones between populations. (j) Splenic and PDA-infiltrating iαβTs, CD4 + T cells, and CD8 + T cells were tested for expression of T-bet. (k) Splenic and orthotopic PDA-infiltrating iαβTs were tested for co-expression of IFNγ and IL-17A. (l) PDA-infiltrating CD3 + IL-17 + cells were gated and tested for the frequency of CD4 + T cells, CD8 + T cells, NKT cells, γδT cells, and iαβTs. (m) Splenic and PDA-infiltrating iαβTs were cultured in vitro for 24h and cell culture supernatant was harvested and assayed for IL-17, IFNγ, and IL-10 (n=5/group). Flow cytometry experiments were repeated more than 4 times with similar results (n=5 mice for each replicate experiment; *p

    Journal: Cancer discovery

    Article Title: Innate αβ T cells Mediate Anti-tumor Immunity by Orchestrating Immunogenic Macrophage Programming

    doi: 10.1158/2159-8290.CD-19-0161

    Figure Lengend Snippet: iαβTs infiltrating PDA are phenotypically distinct. WT mice bearing orthotopic KPC tumors were sacrificed on day 21. (a-g) Splenic and PDA-infiltrating iαβTs were tested for expression of (a) JAML, (b) CD107a, (c) CTLA-4, (d) TIM-3, (e) PD-1, (f) CD39, (g) CD40L, LAG-3, CD73, ICOS, and Dectin-1. (h) Splenic and PDA-infiltrating iαβTs were tested for expression of CD62L. (i ) TCR sequencing of splenic iαβTs and PDA-infiltrating iαβTs, CD4 + and CD8 + T cells was performed in triplicate and assessed for overlapping clones between populations. (j) Splenic and PDA-infiltrating iαβTs, CD4 + T cells, and CD8 + T cells were tested for expression of T-bet. (k) Splenic and orthotopic PDA-infiltrating iαβTs were tested for co-expression of IFNγ and IL-17A. (l) PDA-infiltrating CD3 + IL-17 + cells were gated and tested for the frequency of CD4 + T cells, CD8 + T cells, NKT cells, γδT cells, and iαβTs. (m) Splenic and PDA-infiltrating iαβTs were cultured in vitro for 24h and cell culture supernatant was harvested and assayed for IL-17, IFNγ, and IL-10 (n=5/group). Flow cytometry experiments were repeated more than 4 times with similar results (n=5 mice for each replicate experiment; *p

    Article Snippet: Fluorescently labelled antibodies for CD45 (2D1), TCRαβ (IP26), CD4 (A161A1), CD8 (HIT1a), HLA-DR (L243), TNFα (Mab11), CD11B (ICR F44), IFN-γ (4S.B3), IL-10 (JES3–9D7), ICOS (C398.4A), CD3 (OKT3), CD44 (IM7; all Biolegend) and CD86 (2331FUN-1; BD Biosciences) we used to stain for analysis.

    Techniques: Mouse Assay, Expressing, Sequencing, Clone Assay, Cell Culture, In Vitro, Flow Cytometry

    Levels of exosomes in urine of hypertensive patients. (A) There were no differences among the groups in percent of urinary PL-VAP + /CD31 + , PL-VAP + /CD144 + , and PL-VAP + /CD31 + /CD144 + exosomes. (B) PTC-EMPs were identified using flow cytometry as PL-VAP + /CD31 − /CD144 − as shown in representative fluorescent images. Scale bar =20 µm. (C) Renal vein and systemic levels of PL-VAP + /CD31 − /CD144 − EMPs were not different among the groups, whereas their urinary levels were elevated in both EH and RVH compared to HVs (p

    Journal: Hypertension (Dallas, Tex. : 1979)

    Article Title: Loss of Renal Peritubular Capillaries in Hypertensive Patients is Detectable by Urinary Endothelial Microparticle Levels

    doi: 10.1161/HYPERTENSIONAHA.118.11766

    Figure Lengend Snippet: Levels of exosomes in urine of hypertensive patients. (A) There were no differences among the groups in percent of urinary PL-VAP + /CD31 + , PL-VAP + /CD144 + , and PL-VAP + /CD31 + /CD144 + exosomes. (B) PTC-EMPs were identified using flow cytometry as PL-VAP + /CD31 − /CD144 − as shown in representative fluorescent images. Scale bar =20 µm. (C) Renal vein and systemic levels of PL-VAP + /CD31 − /CD144 − EMPs were not different among the groups, whereas their urinary levels were elevated in both EH and RVH compared to HVs (p

    Article Snippet: In the present study, to measure PTC-EMPs, we compared several endothelial cell markers including PL-VAP, CD31 and CD144.

    Techniques: Flow Cytometry, Cytometry

    Examples of histologically stained tumor sections. The upper row shows NADH-diaphorase stained tumor sections of an untreated tumor, a fully treated tumor, and a partially treated tumor. Treated tumors were excised at 24 h after PDT. The approximate angle of light incidence during PDT is indicated by the arrows. For the partially treated tumor, the black cross roughly indicates the part of the light beam that was blocked by black paper covering the skin. The middle row shows magnifications of H E, NADH-diaphorase, and CD31 stained sections of an untreated tumor. The fluorescence signal of the CD31 detection is shown in red, while the Hoechst 33342 dye injected 5 min before animal sacrifice is shown in blue. The bottom row shows the same types of images for a treated tumor.

    Journal: Theranostics

    Article Title: Detection of Treatment Success after Photodynamic Therapy Using Dynamic Contrast-Enhanced Magnetic Resonance Imaging

    doi: 10.7150/thno.20418

    Figure Lengend Snippet: Examples of histologically stained tumor sections. The upper row shows NADH-diaphorase stained tumor sections of an untreated tumor, a fully treated tumor, and a partially treated tumor. Treated tumors were excised at 24 h after PDT. The approximate angle of light incidence during PDT is indicated by the arrows. For the partially treated tumor, the black cross roughly indicates the part of the light beam that was blocked by black paper covering the skin. The middle row shows magnifications of H E, NADH-diaphorase, and CD31 stained sections of an untreated tumor. The fluorescence signal of the CD31 detection is shown in red, while the Hoechst 33342 dye injected 5 min before animal sacrifice is shown in blue. The bottom row shows the same types of images for a treated tumor.

    Article Snippet: After fixation in ice cold acetone, sections were briefly air-dried, followed by incubation in blocking buffer (Superblock, Thermo Fischer Scientific, Waltham, MA, USA) for 1 h at room temperature, biotin-conjugated rat anti-mouse CD31 (102503, BioLegend, San Diego, CA, USA, dilution 1:250) overnight at 4 °C, and DyLight 649-conjugated streptavidin (405224, BioLegend, dilution 1:100) for 1 h at 20 °C.

    Techniques: Staining, Fluorescence, Injection

    The pY747 peptide inhibits VEGFR2-induced angiogenesis in ex vivo and in vivo . a ) Inhibition of ex vivo endothelial sprouting by the pY747 peptide. Mouse aortic rings were embedded in matrigel in the presence or absence of 40 ng/mL of VEGF and peptides as indicated. Photographs were taken at three days and the number of endothelial sprouts originating from each ring was determined. b ) Quantification of aortic ring assay as indicated in Fig. 3a . c ) Inhibition of in vivo angiogenesis by pY747 peptide. Results of matrigel plug angiogenesis assay are shown. The indicated peptides at 200 µM concentration were mixed with growth factor-reduced matrigel containing VEGF (500 ng/mL) and injected subcutaneously into wild type mice. Seven days later, the matrigel implants were removed, sectioned, and blood vessels were stained with CD31 Ab (red) and nuclei with DAPI (blue). Vessel area was determined using ImagePro. d ) Quantified results of matrigel plug assay as indicated in Fig. 3c .

    Journal: PLoS ONE

    Article Title: Integrin ?3 Crosstalk with VEGFR Accommodating Tyrosine Phosphorylation as a Regulatory Switch

    doi: 10.1371/journal.pone.0031071

    Figure Lengend Snippet: The pY747 peptide inhibits VEGFR2-induced angiogenesis in ex vivo and in vivo . a ) Inhibition of ex vivo endothelial sprouting by the pY747 peptide. Mouse aortic rings were embedded in matrigel in the presence or absence of 40 ng/mL of VEGF and peptides as indicated. Photographs were taken at three days and the number of endothelial sprouts originating from each ring was determined. b ) Quantification of aortic ring assay as indicated in Fig. 3a . c ) Inhibition of in vivo angiogenesis by pY747 peptide. Results of matrigel plug angiogenesis assay are shown. The indicated peptides at 200 µM concentration were mixed with growth factor-reduced matrigel containing VEGF (500 ng/mL) and injected subcutaneously into wild type mice. Seven days later, the matrigel implants were removed, sectioned, and blood vessels were stained with CD31 Ab (red) and nuclei with DAPI (blue). Vessel area was determined using ImagePro. d ) Quantified results of matrigel plug assay as indicated in Fig. 3c .

    Article Snippet: Sections were fixed with 4% paraformaldehyde, incubated with Rat anti-mouse CD31 (BioLegend), and exposed to anti-rat Alexa Fluor568 (Invitrogen).

    Techniques: Ex Vivo, In Vivo, Inhibition, Aortic Ring Assay, Angiogenesis Assay, Concentration Assay, Injection, Mouse Assay, Staining, Matrigel Assay