edta  (Roche)


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    Structured Review

    Roche edta
    Cytokeratin 8 and CD3γ copy numbers in intestinal epithelial cells. A) FACS analysis of T cell contamination. Positive selection of IEC was performed using CD326 Microbeads followed by staining using T-cell receptor antibodies and Pacific Blue Annexin V to detect apoptotic cells. Panel A1 shows an example of IEC isolated using the enzyme based protocol, panel A2 using the <t>EDTA/DTT</t> based protocol. T-cell receptor positive cells are located in quadrant Q1 and Q2. Analysis of absolute mRNA copy numbers of cytokeratin 8 and CD3γ in intestinal epithelial cells separated by positive selection using CD326 Microbeads cells. B) Expression of mRNA copy numbers of Cytokeratin 8 and CD3γ. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p
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    Images

    1) Product Images from "DNA Methylation Analysis in the Intestinal Epithelium--Effect of Cell Separation on Gene Expression and Methylation Profile"

    Article Title: DNA Methylation Analysis in the Intestinal Epithelium--Effect of Cell Separation on Gene Expression and Methylation Profile

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0055636

    Cytokeratin 8 and CD3γ copy numbers in intestinal epithelial cells. A) FACS analysis of T cell contamination. Positive selection of IEC was performed using CD326 Microbeads followed by staining using T-cell receptor antibodies and Pacific Blue Annexin V to detect apoptotic cells. Panel A1 shows an example of IEC isolated using the enzyme based protocol, panel A2 using the EDTA/DTT based protocol. T-cell receptor positive cells are located in quadrant Q1 and Q2. Analysis of absolute mRNA copy numbers of cytokeratin 8 and CD3γ in intestinal epithelial cells separated by positive selection using CD326 Microbeads cells. B) Expression of mRNA copy numbers of Cytokeratin 8 and CD3γ. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p
    Figure Legend Snippet: Cytokeratin 8 and CD3γ copy numbers in intestinal epithelial cells. A) FACS analysis of T cell contamination. Positive selection of IEC was performed using CD326 Microbeads followed by staining using T-cell receptor antibodies and Pacific Blue Annexin V to detect apoptotic cells. Panel A1 shows an example of IEC isolated using the enzyme based protocol, panel A2 using the EDTA/DTT based protocol. T-cell receptor positive cells are located in quadrant Q1 and Q2. Analysis of absolute mRNA copy numbers of cytokeratin 8 and CD3γ in intestinal epithelial cells separated by positive selection using CD326 Microbeads cells. B) Expression of mRNA copy numbers of Cytokeratin 8 and CD3γ. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p

    Techniques Used: FACS, Selection, Staining, Isolation, Expressing, Whisker Assay

    Influence of isolation methods on gene expression profiles. A) Differentially regulated genes in IEC isolated with EDTA/DTT compared to enzymatic release. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p
    Figure Legend Snippet: Influence of isolation methods on gene expression profiles. A) Differentially regulated genes in IEC isolated with EDTA/DTT compared to enzymatic release. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p

    Techniques Used: Isolation, Expressing, Whisker Assay

    2) Product Images from "Hedgehog Signaling Pathway Is Active in GBM with GLI1 mRNA Expression Showing a Single Continuous Distribution Rather than Discrete High/Low Clusters"

    Article Title: Hedgehog Signaling Pathway Is Active in GBM with GLI1 mRNA Expression Showing a Single Continuous Distribution Rather than Discrete High/Low Clusters

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0116390

    A, cell cycle arrest in GBM neurospheres with vismodegib treatment in vitro a) A49910, b), B0027, c) B0043, d) B0051 and e) M45481. B , Number of apoptotic cells (annexin V positive) in GBM neurospheres treated either with DMSO or with 50 μM vismodegib.
    Figure Legend Snippet: A, cell cycle arrest in GBM neurospheres with vismodegib treatment in vitro a) A49910, b), B0027, c) B0043, d) B0051 and e) M45481. B , Number of apoptotic cells (annexin V positive) in GBM neurospheres treated either with DMSO or with 50 μM vismodegib.

    Techniques Used: In Vitro

    Log scale distribution of GLI1 mRNA expression in a) already published MB cases, along with 1 new case of MB from our repository b) distribution of GLI1 mRNA expression (RNA-Seq data) of the TCGA-GBM sub-cohort (N = 149), c) distribution of GLI1 mRNA expression in NIBMG-GBM cases (N = 19), d) distribution of GLI1 mRNA expression in GBM patient-derived early passage neurospheres (N = 6) and e) comparison of median GLI1 mRNA expression levels of high-Hh-MB (N = 13), low-Hh-MB (N-44), NIBMG-GBM (N = 19) and GBM patient-derived neurospheres (N = 6).
    Figure Legend Snippet: Log scale distribution of GLI1 mRNA expression in a) already published MB cases, along with 1 new case of MB from our repository b) distribution of GLI1 mRNA expression (RNA-Seq data) of the TCGA-GBM sub-cohort (N = 149), c) distribution of GLI1 mRNA expression in NIBMG-GBM cases (N = 19), d) distribution of GLI1 mRNA expression in GBM patient-derived early passage neurospheres (N = 6) and e) comparison of median GLI1 mRNA expression levels of high-Hh-MB (N = 13), low-Hh-MB (N-44), NIBMG-GBM (N = 19) and GBM patient-derived neurospheres (N = 6).

    Techniques Used: Expressing, RNA Sequencing Assay, Derivative Assay

    Ligand driven up-regulation and vismodegib driven down-regulation of GLI1 mRNA expressions in 5 GBM neurospheres a) A49910, b), B0027, c) B0043, d) B0051 and e) M45481 (* p-value
    Figure Legend Snippet: Ligand driven up-regulation and vismodegib driven down-regulation of GLI1 mRNA expressions in 5 GBM neurospheres a) A49910, b), B0027, c) B0043, d) B0051 and e) M45481 (* p-value

    Techniques Used:

    3) Product Images from "Increase in Cardiac Ischemia-Reperfusion Injuries in Opa1+/- Mouse Model"

    Article Title: Increase in Cardiac Ischemia-Reperfusion Injuries in Opa1+/- Mouse Model

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0164066

    Calcium transients and SR calcium load in Opa1 +/- isolated left-ventricular cardiomyocytes. A : Typical calcium transients recorded under field stimulation at 1Hz in WT (black) and Opa1 +/- (red) isolated left-ventricular cardiomyocytes using fluo-4 calcium dye. B : Mean values of peak calcium transients (WT, n = 32 cells and 4 animals, vs . Opa1 +/- , n = 43 cells and 5 animals; *p
    Figure Legend Snippet: Calcium transients and SR calcium load in Opa1 +/- isolated left-ventricular cardiomyocytes. A : Typical calcium transients recorded under field stimulation at 1Hz in WT (black) and Opa1 +/- (red) isolated left-ventricular cardiomyocytes using fluo-4 calcium dye. B : Mean values of peak calcium transients (WT, n = 32 cells and 4 animals, vs . Opa1 +/- , n = 43 cells and 5 animals; *p

    Techniques Used: Isolation

    Opa1 +/- left-ventricular cardiomyocyte AP. A : Typical AP recorded using whole-cell patch-clamp technique with current clamp at 1Hz in WT (black) and Opa1 +/- (red) isolated left-ventricular cardiomyocytes. B : Mean values of AP duration at different percentages of AP repolarization (WT: n = 12 cells and 3 animals vs . Opa1 +/- : n = 12 cells and 4 animals; *p
    Figure Legend Snippet: Opa1 +/- left-ventricular cardiomyocyte AP. A : Typical AP recorded using whole-cell patch-clamp technique with current clamp at 1Hz in WT (black) and Opa1 +/- (red) isolated left-ventricular cardiomyocytes. B : Mean values of AP duration at different percentages of AP repolarization (WT: n = 12 cells and 3 animals vs . Opa1 +/- : n = 12 cells and 4 animals; *p

    Techniques Used: Patch Clamp, Isolation

    Dynamic mitochondrial Ca 2+ movements during APs recorded using Rhod-2 in conjunction with a whole-cell patch-clamp technique. A : Typical rhod-2 signal during a steady-state AP recorded at 1Hz in WT (black) and Opa1 +/- (red) isolated left-ventricular cardiomyocytes. B : Mean values of peak rhod-2 signal. C : Rate of rise. D : Decay time constant in WT (n = 12 cells and 3 animals) and Opa1 +/- cells (n = 12 cells and 4 animals; *p
    Figure Legend Snippet: Dynamic mitochondrial Ca 2+ movements during APs recorded using Rhod-2 in conjunction with a whole-cell patch-clamp technique. A : Typical rhod-2 signal during a steady-state AP recorded at 1Hz in WT (black) and Opa1 +/- (red) isolated left-ventricular cardiomyocytes. B : Mean values of peak rhod-2 signal. C : Rate of rise. D : Decay time constant in WT (n = 12 cells and 3 animals) and Opa1 +/- cells (n = 12 cells and 4 animals; *p

    Techniques Used: Patch Clamp, Isolation

    4) Product Images from "Dickkopf-3 links HSF1 and YAP/TAZ signalling to control aggressive behaviours in cancer-associated fibroblasts"

    Article Title: Dickkopf-3 links HSF1 and YAP/TAZ signalling to control aggressive behaviours in cancer-associated fibroblasts

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07987-0

    DKK3 is upregulated in the stroma of breast, colon and ovarian cancers. a Tukey boxplots showing z-score values of DKK3 mRNA expression in normal and cancerous stroma from breast, colorectal and ovarian cancers (Breast: normal, n = 6; cancer, n = 53. Colon: normal, n = 4; cancer, n = 13. Ovary: normal, n = 8; cancer, n = 31). b Representative images of DKK3 staining in breast, colorectal and ovarian cancers and normal tissues. Scale bar, 100 µm. c Tukey boxplots showing quantification of DKK3 staining (Histoscore) in breast, colorectal and ovarian cancers and normal tissue counterparts (Breast: normal/adjacent, n = 9; cancer, n = 109. Colon: normal/adjacent, n = 14; cancer, n = 107. Ovary: normal/adjacent, n = 8; cancer, n = 138). d Tukey boxplots showing DKK3 Histoscore in non-invasive breast cancers (Stage 1 2), invasive breast-cancers (Stage 3 4) and normal tissue counterparts. Left graph shows all cancers irrespective of their subtype (normal, n = 9; Stage 1 2, n = 74; Stage 3 4, n = 74). Middle graph shows ER-negative breast cancers (normal, n = 9; Stage 1 2, n = 40; Stage 3 4, n = 40). Right graphs shows ER-positive breast cancers (normal, n = 9; Stage 1 2, n = 21; Stage 3 4, n = 21). e Disease-free survival of breast cancer patients stratified on stromal DKK3 gene expression (GSE9014, ER-negative patients). f Images show DKK3 (green), vimentin (VIM; red) and DAPI (blue) staining of two representative human breast cancer tissues. Scale bar, 50 µm. g Tukey boxplot shows Dkk3 mRNA expression levels (relative to Gapdh ) in Cancer cells (Epcam + ), immune cells (Cd45 + ), endothelial cells (Cd31 + ) and fibroblasts (Pdgfra + ) from MMTV-PyMT tumorus ( n = 4). h Graphs show correlations between the expression of DKK3 and ACTA2 , FAP and COL1A2 in normal and cancerous stroma from mammary gland (GSE9014), colorectal (GSE35602) and ovarian (GSE40595) human tissues. Pearson correlation coefficient (r) is shown. Each dot represents z-score values from individual patients. i Kaplan–Meier curves of recurrence-free survival, disease-specific survival and progression-free survival of ER-negative breast cancer, colorectal cancer and ovarian cancer patients, respectively, based on DKK3 and DKK2 gene expression. Where indicated, individual p values are shown; alternatively the following symbols were used to describe statistical significance: * P
    Figure Legend Snippet: DKK3 is upregulated in the stroma of breast, colon and ovarian cancers. a Tukey boxplots showing z-score values of DKK3 mRNA expression in normal and cancerous stroma from breast, colorectal and ovarian cancers (Breast: normal, n = 6; cancer, n = 53. Colon: normal, n = 4; cancer, n = 13. Ovary: normal, n = 8; cancer, n = 31). b Representative images of DKK3 staining in breast, colorectal and ovarian cancers and normal tissues. Scale bar, 100 µm. c Tukey boxplots showing quantification of DKK3 staining (Histoscore) in breast, colorectal and ovarian cancers and normal tissue counterparts (Breast: normal/adjacent, n = 9; cancer, n = 109. Colon: normal/adjacent, n = 14; cancer, n = 107. Ovary: normal/adjacent, n = 8; cancer, n = 138). d Tukey boxplots showing DKK3 Histoscore in non-invasive breast cancers (Stage 1 2), invasive breast-cancers (Stage 3 4) and normal tissue counterparts. Left graph shows all cancers irrespective of their subtype (normal, n = 9; Stage 1 2, n = 74; Stage 3 4, n = 74). Middle graph shows ER-negative breast cancers (normal, n = 9; Stage 1 2, n = 40; Stage 3 4, n = 40). Right graphs shows ER-positive breast cancers (normal, n = 9; Stage 1 2, n = 21; Stage 3 4, n = 21). e Disease-free survival of breast cancer patients stratified on stromal DKK3 gene expression (GSE9014, ER-negative patients). f Images show DKK3 (green), vimentin (VIM; red) and DAPI (blue) staining of two representative human breast cancer tissues. Scale bar, 50 µm. g Tukey boxplot shows Dkk3 mRNA expression levels (relative to Gapdh ) in Cancer cells (Epcam + ), immune cells (Cd45 + ), endothelial cells (Cd31 + ) and fibroblasts (Pdgfra + ) from MMTV-PyMT tumorus ( n = 4). h Graphs show correlations between the expression of DKK3 and ACTA2 , FAP and COL1A2 in normal and cancerous stroma from mammary gland (GSE9014), colorectal (GSE35602) and ovarian (GSE40595) human tissues. Pearson correlation coefficient (r) is shown. Each dot represents z-score values from individual patients. i Kaplan–Meier curves of recurrence-free survival, disease-specific survival and progression-free survival of ER-negative breast cancer, colorectal cancer and ovarian cancer patients, respectively, based on DKK3 and DKK2 gene expression. Where indicated, individual p values are shown; alternatively the following symbols were used to describe statistical significance: * P

    Techniques Used: Expressing, Staining

    5) Product Images from "Adoptive Transfer of Immunomodulatory M2 Macrophages Prevents Type 1 Diabetes in NOD Mice"

    Article Title: Adoptive Transfer of Immunomodulatory M2 Macrophages Prevents Type 1 Diabetes in NOD Mice

    Journal: Diabetes

    doi: 10.2337/db11-1635

    M2r macrophages suppress ex vivo T-cell activity. M2r or M0 macrophages (2.5 × 10 6 ) were intraperitoneally injected into 12–16-week-old NOD-BDC2.5 mice. A : PLNs were dissected after 1 week, and lymphocytes were restimulated with BDC2.5 mimotope for 72 h to induce proliferation. Readout was CPM . B : PLNs were dissected after 1 week, and lymphocyte subsets were analyzed by flow cytometry for cell numbers (percent and absolute values). C : Activation status of CD4 + subset assessed by CD44 and CD62L expression. D : Pancreata were dissected after 1 week, and lymphocyte subsets were analyzed by flow cytometry for cell numbers (percent and absolute values). E : M2r macrophages (2–3 × 10 6 ) or control (PBS) were intraperitoneally injected into 12–13-week-old NOD-FoxP3-GFP mice, PLNs were dissected after 1 week, and T-cell subset numbers were analyzed. The data from A – D represent two independent experiments ( n = 4). The data in E represent pooled data from four independent experiments. Error bars are presented in SEM. * P
    Figure Legend Snippet: M2r macrophages suppress ex vivo T-cell activity. M2r or M0 macrophages (2.5 × 10 6 ) were intraperitoneally injected into 12–16-week-old NOD-BDC2.5 mice. A : PLNs were dissected after 1 week, and lymphocytes were restimulated with BDC2.5 mimotope for 72 h to induce proliferation. Readout was CPM . B : PLNs were dissected after 1 week, and lymphocyte subsets were analyzed by flow cytometry for cell numbers (percent and absolute values). C : Activation status of CD4 + subset assessed by CD44 and CD62L expression. D : Pancreata were dissected after 1 week, and lymphocyte subsets were analyzed by flow cytometry for cell numbers (percent and absolute values). E : M2r macrophages (2–3 × 10 6 ) or control (PBS) were intraperitoneally injected into 12–13-week-old NOD-FoxP3-GFP mice, PLNs were dissected after 1 week, and T-cell subset numbers were analyzed. The data from A – D represent two independent experiments ( n = 4). The data in E represent pooled data from four independent experiments. Error bars are presented in SEM. * P

    Techniques Used: Ex Vivo, Activity Assay, Injection, Mouse Assay, Flow Cytometry, Cytometry, Activation Assay, Expressing

    Macrophages predominantly migrate to the pancreas and PLN. A : NOD mice (10–12 weeks of age) received 3 × 10 6 DiR-labeled M2r or M0 macrophages intraperitoneally. Mice were anesthetized prior to anterior imaging after 2 h and 1, 3, 6, and 8 days post–macrophage injection. Bright yellow or dark red represents high or low photon counts, respectively, and white arrows indicate injection sites. Two individual mice were analyzed, and the data represent two individual experiments. B : After transfer, the liver, kidney, spleen, pancreas, and PLNs were dissected after 3 days post–macrophage injection. The image is representative of eight individual mice (four treated with M0 and four with M2r). C : Photon count from each organ in B was quantified, and control organ photon count was subtracted to obtain the real macrophage emission. Error bars are presented in SEM. D : NOD mice received 2 × 10 6 DiI-labeled M2r macrophages (red) intraperitoneally, and pancreata were dissected at day 3 and stained for CD11b (green) and DAPI (blue). The data are representative of four different mice. (A high-quality digital representation of this figure is available in the online issue.)
    Figure Legend Snippet: Macrophages predominantly migrate to the pancreas and PLN. A : NOD mice (10–12 weeks of age) received 3 × 10 6 DiR-labeled M2r or M0 macrophages intraperitoneally. Mice were anesthetized prior to anterior imaging after 2 h and 1, 3, 6, and 8 days post–macrophage injection. Bright yellow or dark red represents high or low photon counts, respectively, and white arrows indicate injection sites. Two individual mice were analyzed, and the data represent two individual experiments. B : After transfer, the liver, kidney, spleen, pancreas, and PLNs were dissected after 3 days post–macrophage injection. The image is representative of eight individual mice (four treated with M0 and four with M2r). C : Photon count from each organ in B was quantified, and control organ photon count was subtracted to obtain the real macrophage emission. Error bars are presented in SEM. D : NOD mice received 2 × 10 6 DiI-labeled M2r macrophages (red) intraperitoneally, and pancreata were dissected at day 3 and stained for CD11b (green) and DAPI (blue). The data are representative of four different mice. (A high-quality digital representation of this figure is available in the online issue.)

    Techniques Used: Mouse Assay, Labeling, Imaging, Injection, Staining

    M2r macrophages retain an M2 signature after secondary proinflammatory stimulation both in vitro and in vivo. A : Schematic experimental set-up. Primary activation with M0-, M2r-, or M1-inducing stimuli was for 24 h, and secondary activation for an additional 24 h with LPS/IFNγ (48 h). B : IL-10 production assessed by ELISA in secondary-activated macrophages. C : Flow cytometry analyses of CD86 and PD-L2 expression on primary- and secondary-activated macrophages. Histograms and mean fluorescence intensity analyses are depicted. MFI, mean fluorescence intensity. D : Selected gene expression assessed by RT-PCR after primary and secondary activations of macrophages. E : Ex vivo analyses of CD86 and PD-L2 on CD11b + DiD + macrophages recovered from pancreata ( left ) or PLNs ( right ). Histograms and percentage of cells depicted. The results are representative of two independent experiments ( n = 4). * P
    Figure Legend Snippet: M2r macrophages retain an M2 signature after secondary proinflammatory stimulation both in vitro and in vivo. A : Schematic experimental set-up. Primary activation with M0-, M2r-, or M1-inducing stimuli was for 24 h, and secondary activation for an additional 24 h with LPS/IFNγ (48 h). B : IL-10 production assessed by ELISA in secondary-activated macrophages. C : Flow cytometry analyses of CD86 and PD-L2 expression on primary- and secondary-activated macrophages. Histograms and mean fluorescence intensity analyses are depicted. MFI, mean fluorescence intensity. D : Selected gene expression assessed by RT-PCR after primary and secondary activations of macrophages. E : Ex vivo analyses of CD86 and PD-L2 on CD11b + DiD + macrophages recovered from pancreata ( left ) or PLNs ( right ). Histograms and percentage of cells depicted. The results are representative of two independent experiments ( n = 4). * P

    Techniques Used: In Vitro, In Vivo, Activation Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Expressing, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Ex Vivo

    6) Product Images from "DNA Methylation Analysis in the Intestinal Epithelium--Effect of Cell Separation on Gene Expression and Methylation Profile"

    Article Title: DNA Methylation Analysis in the Intestinal Epithelium--Effect of Cell Separation on Gene Expression and Methylation Profile

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0055636

    Cytokeratin 8 and CD3γ copy numbers in intestinal epithelial cells. A) FACS analysis of T cell contamination. Positive selection of IEC was performed using CD326 Microbeads followed by staining using T-cell receptor antibodies and Pacific Blue Annexin V to detect apoptotic cells. Panel A1 shows an example of IEC isolated using the enzyme based protocol, panel A2 using the EDTA/DTT based protocol. T-cell receptor positive cells are located in quadrant Q1 and Q2. Analysis of absolute mRNA copy numbers of cytokeratin 8 and CD3γ in intestinal epithelial cells separated by positive selection using CD326 Microbeads cells. B) Expression of mRNA copy numbers of Cytokeratin 8 and CD3γ. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p
    Figure Legend Snippet: Cytokeratin 8 and CD3γ copy numbers in intestinal epithelial cells. A) FACS analysis of T cell contamination. Positive selection of IEC was performed using CD326 Microbeads followed by staining using T-cell receptor antibodies and Pacific Blue Annexin V to detect apoptotic cells. Panel A1 shows an example of IEC isolated using the enzyme based protocol, panel A2 using the EDTA/DTT based protocol. T-cell receptor positive cells are located in quadrant Q1 and Q2. Analysis of absolute mRNA copy numbers of cytokeratin 8 and CD3γ in intestinal epithelial cells separated by positive selection using CD326 Microbeads cells. B) Expression of mRNA copy numbers of Cytokeratin 8 and CD3γ. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p

    Techniques Used: FACS, Selection, Staining, Isolation, Expressing, Whisker Assay

    Influence of isolation methods on gene expression profiles. A) Differentially regulated genes in IEC isolated with EDTA/DTT compared to enzymatic release. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p
    Figure Legend Snippet: Influence of isolation methods on gene expression profiles. A) Differentially regulated genes in IEC isolated with EDTA/DTT compared to enzymatic release. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p

    Techniques Used: Isolation, Expressing, Whisker Assay

    7) Product Images from "Thy-1 Deficiency Augments Bone Loss in Obesity by Affecting Bone Formation and Resorption"

    Article Title: Thy-1 Deficiency Augments Bone Loss in Obesity by Affecting Bone Formation and Resorption

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2018.00127

    Lack of Thy-1 reduces osteoblast differentiation as well as bone formation and increases osteoclast differentiation and bone resorption in obese mice. Wildtype (WT) and Thy-1 −/− (KO) mice were fed with a high fat diet for 18 weeks (HFD). (A–J) Bone formation and (K-O) bone resorption were analyzed. The (A) osteoblast number per bone perimeter (Ob.N/B.Pm), (B) osteoblast surface per bone surface (Ob.S/BS) were analyzed using histology methods. Gene expression of the osteogenic markers (C) runt-related transcription factor 2 ( Runx2 ), (D) and alkaline phosphatase ( Tnalp ) was analyzed by RT-PCR technique. (E) The serum concentration of total procollagen type 1 amino-terminal propeptide (P1NP) was analyzed using ELISA technique and the (F) osteoid surface per bone perimeter (Osteid.S/B.Pm) by histology. (G) Representative sections of von Kossa/van Gieson staining of bone (black) and cartilage (dense red area close to the bone). (H) The bone formation rate per bone surface (BFR/BS) as well as (I) mineral apposition rate (MAR) were determined by histomorphometric analysis (double calcein labeling). (J) Representative images of calcein labeling (green). (K) Osteoclast number per bone perimeter (Oc.N/B.Pm) and (L) osteoclast surface per bone surface (Oc.S/BS) were analyzed by staining of tartrate resistant acid phosphatase (TRAP). (M) Representative images of TRAP staining (red spots and black arrows = TRAP-positive cell/osteoclast). (N) Serum concentration of the bone resorption marker carboxy-terminal collagen crosslinks (CTX) was measured by ELISA technique. (O) Gene expression of Trap was evaluated via RT-PCR. Each point represents one mouse and median ± SD is presented. Asterisks denote significance level of ∗ P
    Figure Legend Snippet: Lack of Thy-1 reduces osteoblast differentiation as well as bone formation and increases osteoclast differentiation and bone resorption in obese mice. Wildtype (WT) and Thy-1 −/− (KO) mice were fed with a high fat diet for 18 weeks (HFD). (A–J) Bone formation and (K-O) bone resorption were analyzed. The (A) osteoblast number per bone perimeter (Ob.N/B.Pm), (B) osteoblast surface per bone surface (Ob.S/BS) were analyzed using histology methods. Gene expression of the osteogenic markers (C) runt-related transcription factor 2 ( Runx2 ), (D) and alkaline phosphatase ( Tnalp ) was analyzed by RT-PCR technique. (E) The serum concentration of total procollagen type 1 amino-terminal propeptide (P1NP) was analyzed using ELISA technique and the (F) osteoid surface per bone perimeter (Osteid.S/B.Pm) by histology. (G) Representative sections of von Kossa/van Gieson staining of bone (black) and cartilage (dense red area close to the bone). (H) The bone formation rate per bone surface (BFR/BS) as well as (I) mineral apposition rate (MAR) were determined by histomorphometric analysis (double calcein labeling). (J) Representative images of calcein labeling (green). (K) Osteoclast number per bone perimeter (Oc.N/B.Pm) and (L) osteoclast surface per bone surface (Oc.S/BS) were analyzed by staining of tartrate resistant acid phosphatase (TRAP). (M) Representative images of TRAP staining (red spots and black arrows = TRAP-positive cell/osteoclast). (N) Serum concentration of the bone resorption marker carboxy-terminal collagen crosslinks (CTX) was measured by ELISA technique. (O) Gene expression of Trap was evaluated via RT-PCR. Each point represents one mouse and median ± SD is presented. Asterisks denote significance level of ∗ P

    Techniques Used: Mouse Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Labeling, Marker

    Obese Thy-1 −/− mice display decreased trabecular bone mass while cortical bone mass and biomechanical properties are unaltered. Wildtype (WT) and Thy-1 −/− (KO) mice were fed with a high fat diet for 18 weeks (HFD). As control group, WT mice were fed a standard chow (CHOW) for the same time period. (A) The body weight of standard and HFD fed WT and KO mice over 18 weeks. Hashtags denote significance level of # P
    Figure Legend Snippet: Obese Thy-1 −/− mice display decreased trabecular bone mass while cortical bone mass and biomechanical properties are unaltered. Wildtype (WT) and Thy-1 −/− (KO) mice were fed with a high fat diet for 18 weeks (HFD). As control group, WT mice were fed a standard chow (CHOW) for the same time period. (A) The body weight of standard and HFD fed WT and KO mice over 18 weeks. Hashtags denote significance level of # P

    Techniques Used: Mouse Assay

    Lack of Thy-1 promotes obesity mediated inflammation. Wildtype (WT) and Thy-1 −/− (KO) mice were fed with a high fat diet for 18 weeks (HFD). (A) Number of adipocytes per total area (N.Adipo/Tt.Ar) and (B) adipocyte area (Adipo.Ar) of adipocytes were analyzed by histology technique. (C) Gene expression of the fat marker fatty-acid-binding protein ( Fabp ) in bone was assessed by RT-PCR. (D) Fat volume (FV/TV) in the femoral medullary cavity was analyzed by osmium tetroxide staining. (E) Representative 3D-images of the fat volume of whole femur. (F,G) Gene expression of the pro-inflammatory markers tumor necrosis factor α ( Tnfα ) and interleukin 6 ( Il6 ). (H) Osteoclast precursor cells from WT mice were cultured ex vivo without (w/o) and with TNFα and osteoclastogenesis was detected via staining for tartrate resistant acid phosphatase (TRAP; giant, multinucleated, red cells = osteoclasts; indicated by arrows). Asterisks denote significance level of ∗ P
    Figure Legend Snippet: Lack of Thy-1 promotes obesity mediated inflammation. Wildtype (WT) and Thy-1 −/− (KO) mice were fed with a high fat diet for 18 weeks (HFD). (A) Number of adipocytes per total area (N.Adipo/Tt.Ar) and (B) adipocyte area (Adipo.Ar) of adipocytes were analyzed by histology technique. (C) Gene expression of the fat marker fatty-acid-binding protein ( Fabp ) in bone was assessed by RT-PCR. (D) Fat volume (FV/TV) in the femoral medullary cavity was analyzed by osmium tetroxide staining. (E) Representative 3D-images of the fat volume of whole femur. (F,G) Gene expression of the pro-inflammatory markers tumor necrosis factor α ( Tnfα ) and interleukin 6 ( Il6 ). (H) Osteoclast precursor cells from WT mice were cultured ex vivo without (w/o) and with TNFα and osteoclastogenesis was detected via staining for tartrate resistant acid phosphatase (TRAP; giant, multinucleated, red cells = osteoclasts; indicated by arrows). Asterisks denote significance level of ∗ P

    Techniques Used: Mouse Assay, Expressing, Marker, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Staining, Cell Culture, Ex Vivo

    Thy-1 −/− in obesity does not alter the Wnt and YAZ/TAZ pathway. Wildtype (WT) and Thy-1 −/− (KO) mice were fed with a high fat diet for a time period of 18 weeks (HFD). Gene expression of the Wnt pathway inhibitors (A) dickkopf-1 ( Dkk-1 ) and (B) sclerostin ( Sost ) and (C,D) their serum concentrations were evaluated by RT-PCR and ELISA technique, respectively. Gene expression of Wnt ligands such as (E) Wnt5a , (F) 11 , (G) 3a , and (H) 10b in bone was analyzed by RT-PCR. MSCs from WT and Thy-1 −/− mice were treated with TNFα and expression of Yap and Taz of the hippo signaling were investigated. Statistical analysis was performed by (A–H) Student’s t -test and by (I,J) 2-way ANOVA.
    Figure Legend Snippet: Thy-1 −/− in obesity does not alter the Wnt and YAZ/TAZ pathway. Wildtype (WT) and Thy-1 −/− (KO) mice were fed with a high fat diet for a time period of 18 weeks (HFD). Gene expression of the Wnt pathway inhibitors (A) dickkopf-1 ( Dkk-1 ) and (B) sclerostin ( Sost ) and (C,D) their serum concentrations were evaluated by RT-PCR and ELISA technique, respectively. Gene expression of Wnt ligands such as (E) Wnt5a , (F) 11 , (G) 3a , and (H) 10b in bone was analyzed by RT-PCR. MSCs from WT and Thy-1 −/− mice were treated with TNFα and expression of Yap and Taz of the hippo signaling were investigated. Statistical analysis was performed by (A–H) Student’s t -test and by (I,J) 2-way ANOVA.

    Techniques Used: Mouse Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Lack of Thy-1 in obese mice alters the gene expression of RANKL, OPG, and CSF1 under inflammatory conditions. Wildtype (WT) and Thy-1 −/− (KO) mice were fed with a high fat diet for a time period of 18 weeks (HFD). (A) Gene expression of receptor activator of NF-κB ligand (RANKL, Tnfsf11 ), (B) its decoy receptor osteoprotegerin (OPG, Tnfrsf11b ), and (C) receptor of Csf1 (Cfs1r) was analyzed in bone. MSCs from WT and KO mice were treated with TNFα for 24 h to mimic an inflammatory environment and gene expression of Tnfsf11 , Tnfrsf11b , and Cfs1 was determined (D–F) . (G) Summary figure of the key findings. In mice, Thy-1 deficiency results in a reduced osteoclastogenesis and increased adipogenesis leading to a decreased bone formation. Adipocytes produce more of the pro-inflammatory cytokine TNFα and the RANKL-OPG ratio is reduced resulting in an elevated osteoclastogenesis and poor bone mass. Each point represents one mouse and median ± SD is presented. Asterisks denote significance level of ∗ P
    Figure Legend Snippet: Lack of Thy-1 in obese mice alters the gene expression of RANKL, OPG, and CSF1 under inflammatory conditions. Wildtype (WT) and Thy-1 −/− (KO) mice were fed with a high fat diet for a time period of 18 weeks (HFD). (A) Gene expression of receptor activator of NF-κB ligand (RANKL, Tnfsf11 ), (B) its decoy receptor osteoprotegerin (OPG, Tnfrsf11b ), and (C) receptor of Csf1 (Cfs1r) was analyzed in bone. MSCs from WT and KO mice were treated with TNFα for 24 h to mimic an inflammatory environment and gene expression of Tnfsf11 , Tnfrsf11b , and Cfs1 was determined (D–F) . (G) Summary figure of the key findings. In mice, Thy-1 deficiency results in a reduced osteoclastogenesis and increased adipogenesis leading to a decreased bone formation. Adipocytes produce more of the pro-inflammatory cytokine TNFα and the RANKL-OPG ratio is reduced resulting in an elevated osteoclastogenesis and poor bone mass. Each point represents one mouse and median ± SD is presented. Asterisks denote significance level of ∗ P

    Techniques Used: Mouse Assay, Expressing

    8) Product Images from "Oncogenic RAS Signaling Promotes Tumor Immunoresistance by Stabilizing PD-L1 mRNA"

    Article Title: Oncogenic RAS Signaling Promotes Tumor Immunoresistance by Stabilizing PD-L1 mRNA

    Journal: Immunity

    doi: 10.1016/j.immuni.2017.11.016

    RAS-ROS-p38 Signaling Controls TTP Activity (A) Histograms represent peak areas from extracted ion chromatograms for non-phosphorylated and phosphorylated peptides corresponding to S52 and S178 phosphosites of mouse TTP. Myc-TTP was immunoprecipitated from CT26 Myc-TTP (tet-ON) cells 1 hr after the indicated treatment. Mean ± SD of technical triplicates. Representative of two independent biological experiments. (B) qPCR analysis of ER-KRAS G12V type II pneumocytes treated in starvation medium for 24 hr. Mean ± SEM of four independent experiments. (C) Representative flow cytometry histograms of PD-L1 surface protein expression in MCF10A ER-ΔMEKK3 cells treated in starvation medium for 1 day or 4 days. Data are representative of two independent experiments. (D) Flow cytometry analysis of PD-L1 surface protein expression on ER-HRAS G12V MCF10A cells (24 hr) and ER-HRAS G12V HKE-3 cells (48 hr) after treatment in starvation medium. Data are representative of biological duplicates. (E) qPCR analysis of CT26 cells at 2 hr or 24 hr after MK2 inhibition with PF 3644022. Mean ± SEM of two independent experiments. (F) Sequence alignments of the conserved phosphosites (highlighted red) targeted by MK2 in mouse ( Mm ) and human ( Hs ) TTP protein. (G) Western blotting of immunoprecipitations from CT26 TTP KO cells harboring tet-ON, WT, or phospho mutant, Myc-TTP constructs. Cells were treated with dox. for 24 hr before the addition of PMA or DMSO for 1 hr. Arrow indicates Myc-TTP. Data are representative of two independent experiments. (H) qPCR analysis of CT26 TTP KO cells harboring tet-ON, WT, or phospho mutant, Myc-TTP constructs, treated with dox or vehicle for 48 hr. Data represent the mean ± SEM of two independent experiments. ∗∗ p
    Figure Legend Snippet: RAS-ROS-p38 Signaling Controls TTP Activity (A) Histograms represent peak areas from extracted ion chromatograms for non-phosphorylated and phosphorylated peptides corresponding to S52 and S178 phosphosites of mouse TTP. Myc-TTP was immunoprecipitated from CT26 Myc-TTP (tet-ON) cells 1 hr after the indicated treatment. Mean ± SD of technical triplicates. Representative of two independent biological experiments. (B) qPCR analysis of ER-KRAS G12V type II pneumocytes treated in starvation medium for 24 hr. Mean ± SEM of four independent experiments. (C) Representative flow cytometry histograms of PD-L1 surface protein expression in MCF10A ER-ΔMEKK3 cells treated in starvation medium for 1 day or 4 days. Data are representative of two independent experiments. (D) Flow cytometry analysis of PD-L1 surface protein expression on ER-HRAS G12V MCF10A cells (24 hr) and ER-HRAS G12V HKE-3 cells (48 hr) after treatment in starvation medium. Data are representative of biological duplicates. (E) qPCR analysis of CT26 cells at 2 hr or 24 hr after MK2 inhibition with PF 3644022. Mean ± SEM of two independent experiments. (F) Sequence alignments of the conserved phosphosites (highlighted red) targeted by MK2 in mouse ( Mm ) and human ( Hs ) TTP protein. (G) Western blotting of immunoprecipitations from CT26 TTP KO cells harboring tet-ON, WT, or phospho mutant, Myc-TTP constructs. Cells were treated with dox. for 24 hr before the addition of PMA or DMSO for 1 hr. Arrow indicates Myc-TTP. Data are representative of two independent experiments. (H) qPCR analysis of CT26 TTP KO cells harboring tet-ON, WT, or phospho mutant, Myc-TTP constructs, treated with dox or vehicle for 48 hr. Data represent the mean ± SEM of two independent experiments. ∗∗ p

    Techniques Used: Activity Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Expressing, Inhibition, Sequencing, Western Blot, Mutagenesis, Construct

    Restoration of Tumor Cell TTP Expression Enhances Anti-tumor Immunity (A) Western blotting analysis of CT26 Myc-TTP tet-ON cells expressing either empty vector or mouse Cd274 cDNA lacking the 3′ UTR (PD-L1 Δ3′ UTR), 24 hr after treatment (Dox., 0.1 μg/mL or 1 μg/mL). Arrow indicates Myc-TTP. Data are representative of two independent experiments. (B) Representative flow cytometry histograms of PD-L1 surface protein expression in CT26 stable cells lines in (A), 72 hr after treatment (Dox., 1 μg/mL). Data are representative of three independent experiments. (C) Tumor growth curves for CT26-derived cell lines subcutaneously transplanted into BALB/c mice (n = 8 per group). (D) Tumor growth curves for MC38-derived cell lines subcutaneously transplanted into C57BL/6 mice (n = 6 per group). X denotes the loss of a doxycycline-treated mouse. (E) Tumor growth curves for CT26-derived cell lines subcutaneously transplanted into nu/nu mice (n = 6 per group). (F) Tumor growth curves for CT26-derived cell lines subcutaneously transplanted into BALB/c mice (n = 4–5 per group). For (C)–(F), data represent the mean ± SEM from individual experiments. ∗∗ p
    Figure Legend Snippet: Restoration of Tumor Cell TTP Expression Enhances Anti-tumor Immunity (A) Western blotting analysis of CT26 Myc-TTP tet-ON cells expressing either empty vector or mouse Cd274 cDNA lacking the 3′ UTR (PD-L1 Δ3′ UTR), 24 hr after treatment (Dox., 0.1 μg/mL or 1 μg/mL). Arrow indicates Myc-TTP. Data are representative of two independent experiments. (B) Representative flow cytometry histograms of PD-L1 surface protein expression in CT26 stable cells lines in (A), 72 hr after treatment (Dox., 1 μg/mL). Data are representative of three independent experiments. (C) Tumor growth curves for CT26-derived cell lines subcutaneously transplanted into BALB/c mice (n = 8 per group). (D) Tumor growth curves for MC38-derived cell lines subcutaneously transplanted into C57BL/6 mice (n = 6 per group). X denotes the loss of a doxycycline-treated mouse. (E) Tumor growth curves for CT26-derived cell lines subcutaneously transplanted into nu/nu mice (n = 6 per group). (F) Tumor growth curves for CT26-derived cell lines subcutaneously transplanted into BALB/c mice (n = 4–5 per group). For (C)–(F), data represent the mean ± SEM from individual experiments. ∗∗ p

    Techniques Used: Expressing, Western Blot, Plasmid Preparation, Flow Cytometry, Cytometry, Derivative Assay, Mouse Assay

    Cell-Intrinsic Upregulation of PD-L1 through Oncogenic RAS Signaling (A) Western blotting analysis of ER-KRAS G12V type II pneumocytes treated with 4-OHT in starvation medium. Phospho-ERK and phospho-AKT was measured over time to monitor RAS pathway activation. Data are representative of two independent experiments. (B) qPCR analysis of ER-KRAS G12V type II pneumocytes treated with 4-OHT or IFN-γ in starvation medium. Mean ± SEM of biological duplicates (n = 2) from the experiment described in (A). (C) Representative flow cytometry histogram of PD-L1 surface protein expression in ER-KRAS G12V type II pneumocytes treated in starvation medium for 48 hr. Data are representative of two independent experiments. (D) Western blotting analysis of RAS signaling following 5 hr treatment with the KRAS G12C inhibitor ARS853. Phospho-ERK and phospho-AKT signal reflect RAS pathway activity. Data are representative of two independent experiments. (E) qPCR analysis following 5 hr treatment with the KRAS G12C inhibitor ARS853 (10 μM). Mean ± SEM of biological duplicates (n = 2) from the experiment described in (D). (F) Flow cytometry analysis of PD-L1 surface protein expression in H358 cells treated with ARS853 (10 μM) for 48 hr. Mean ± SEM of biological triplicates. (G) Flow cytometry analysis of PD-L1 surface protein expression in ER-KRAS G12V type II pneumocytes treated in starvation medium for 24 hr. Mean ± SEM of two independent experiments. (H) qPCR analysis from the experiment described in (G). Mean ± SEM of biological triplicates pooled from two independent experiments. (I) qPCR analysis of H358 cells treated for 24 hr. Mean ± SEM of two independent experiments. (J) qPCR analysis of H358 cells treated with PMA for 3 hr following a 30 min pre-treatment with DMSO or MEK inhibitor. Mean ± SD of two independent experiments. Abbreviations and quantities are as follows: MFI, mean fluorescence intensity; EtOH, ethanol vehicle; 4-OHT, 100 nM; IFN-γ, 20 ng/mL; MEK inhibitor GSK1120212, 25 nM; PI3K inhibitor GDC-0941, 500 nM; PMA, 200 nM. ∗∗∗∗ p
    Figure Legend Snippet: Cell-Intrinsic Upregulation of PD-L1 through Oncogenic RAS Signaling (A) Western blotting analysis of ER-KRAS G12V type II pneumocytes treated with 4-OHT in starvation medium. Phospho-ERK and phospho-AKT was measured over time to monitor RAS pathway activation. Data are representative of two independent experiments. (B) qPCR analysis of ER-KRAS G12V type II pneumocytes treated with 4-OHT or IFN-γ in starvation medium. Mean ± SEM of biological duplicates (n = 2) from the experiment described in (A). (C) Representative flow cytometry histogram of PD-L1 surface protein expression in ER-KRAS G12V type II pneumocytes treated in starvation medium for 48 hr. Data are representative of two independent experiments. (D) Western blotting analysis of RAS signaling following 5 hr treatment with the KRAS G12C inhibitor ARS853. Phospho-ERK and phospho-AKT signal reflect RAS pathway activity. Data are representative of two independent experiments. (E) qPCR analysis following 5 hr treatment with the KRAS G12C inhibitor ARS853 (10 μM). Mean ± SEM of biological duplicates (n = 2) from the experiment described in (D). (F) Flow cytometry analysis of PD-L1 surface protein expression in H358 cells treated with ARS853 (10 μM) for 48 hr. Mean ± SEM of biological triplicates. (G) Flow cytometry analysis of PD-L1 surface protein expression in ER-KRAS G12V type II pneumocytes treated in starvation medium for 24 hr. Mean ± SEM of two independent experiments. (H) qPCR analysis from the experiment described in (G). Mean ± SEM of biological triplicates pooled from two independent experiments. (I) qPCR analysis of H358 cells treated for 24 hr. Mean ± SEM of two independent experiments. (J) qPCR analysis of H358 cells treated with PMA for 3 hr following a 30 min pre-treatment with DMSO or MEK inhibitor. Mean ± SD of two independent experiments. Abbreviations and quantities are as follows: MFI, mean fluorescence intensity; EtOH, ethanol vehicle; 4-OHT, 100 nM; IFN-γ, 20 ng/mL; MEK inhibitor GSK1120212, 25 nM; PI3K inhibitor GDC-0941, 500 nM; PMA, 200 nM. ∗∗∗∗ p

    Techniques Used: Western Blot, Activation Assay, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Expressing, Activity Assay, Fluorescence

    9) Product Images from "Thymocytes trigger self-antigen-controlling pathways in immature medullary thymic epithelial stages"

    Article Title: Thymocytes trigger self-antigen-controlling pathways in immature medullary thymic epithelial stages

    Journal: bioRxiv

    doi: 10.1101/2020.11.29.399923

    The transcriptional and functional properties of mTEC lo are impaired in mTEC ΔMHCII mice. (A) Percentages of CD69 + OTII CD4 + T cells cultured or not with variable numbers of OVA 323-339 -loaded WT or mTEC ΔMHCII mTECs derived of 2 independent experiments (n=2-3 mice per group and experiment). (B) Scatter-plot of gene expression levels (FPKM) of mTEC lo from WT versus mTEC ΔMHCII mice. Genes with fold difference ≥2 and p-adj
    Figure Legend Snippet: The transcriptional and functional properties of mTEC lo are impaired in mTEC ΔMHCII mice. (A) Percentages of CD69 + OTII CD4 + T cells cultured or not with variable numbers of OVA 323-339 -loaded WT or mTEC ΔMHCII mTECs derived of 2 independent experiments (n=2-3 mice per group and experiment). (B) Scatter-plot of gene expression levels (FPKM) of mTEC lo from WT versus mTEC ΔMHCII mice. Genes with fold difference ≥2 and p-adj

    Techniques Used: Functional Assay, Mouse Assay, Cell Culture, Derivative Assay, Expressing

    Highly self-reactive CD4 + thymocytes control mTEC development from an early progenitor stage. (A-D) Flow cytometry profiles and numbers of total TECs (EpCAM + ) (A) , cTECs (UEA-1 - Ly51 hi ), mTECs (UEA-1 - Ly51 lo ) (B) , TEC lo (MHCII lo UEA-1 lo ), cTEC hi (MHCII hi UEA-1 lo ), mTEC lo (MHCII lo UEA-1 hi ) and mTEC hi (MHCII hi UEA-1 hi ) (C) , α6-integrin hi Sca-1 hi TEPC-enriched cells in TEC lo (D) in CD45 neg -enriched cells from RipmOVAxOTII- Rag2 -/- and OTII- Rag2 -/- mice. Data are representative of 4 experiments (n=3 mice per group and experiment). (E) Confocal images of thymic sections from RipmOVAxOTII- Rag2 -/- and OTII- Rag2 -/- mice stained for Aire (green) and Fezf2 (red). 11 and 22 sections derived from 2 RipmOVAxOTII- Rag2 -/- and OTII- Rag2 -/- mice were quantified, respectively. Scale bar, 50 μm. Unfilled, dashed and solid arrowheads indicate Aire + Fezf2 lo , Aire - Fezf2 + and Aire + Fezf2 + cells, respectively. The histogram shows the density of Aire + Fezf2 lo , Aire - Fezf2 + and Aire + Fezf2 + cells. (F,G) Flow cytometry profiles and numbers of Aire - Fezf2 - , Aire - Fezf2 + and Aire + Fezf2 + cells in total mTECs, mTEC lo and mTEC hi (F) and of DCKL1 + cells in Aire - mTECs (G) from RipmOVAxOTII- Rag2 -/- and OTII- Rag2 -/- mice. II Abs: secondary antibodies. Data are representative of 2 independent experiments (n=3-4 mice per group and experiment). Error bars show mean◻±◻SEM, *p
    Figure Legend Snippet: Highly self-reactive CD4 + thymocytes control mTEC development from an early progenitor stage. (A-D) Flow cytometry profiles and numbers of total TECs (EpCAM + ) (A) , cTECs (UEA-1 - Ly51 hi ), mTECs (UEA-1 - Ly51 lo ) (B) , TEC lo (MHCII lo UEA-1 lo ), cTEC hi (MHCII hi UEA-1 lo ), mTEC lo (MHCII lo UEA-1 hi ) and mTEC hi (MHCII hi UEA-1 hi ) (C) , α6-integrin hi Sca-1 hi TEPC-enriched cells in TEC lo (D) in CD45 neg -enriched cells from RipmOVAxOTII- Rag2 -/- and OTII- Rag2 -/- mice. Data are representative of 4 experiments (n=3 mice per group and experiment). (E) Confocal images of thymic sections from RipmOVAxOTII- Rag2 -/- and OTII- Rag2 -/- mice stained for Aire (green) and Fezf2 (red). 11 and 22 sections derived from 2 RipmOVAxOTII- Rag2 -/- and OTII- Rag2 -/- mice were quantified, respectively. Scale bar, 50 μm. Unfilled, dashed and solid arrowheads indicate Aire + Fezf2 lo , Aire - Fezf2 + and Aire + Fezf2 + cells, respectively. The histogram shows the density of Aire + Fezf2 lo , Aire - Fezf2 + and Aire + Fezf2 + cells. (F,G) Flow cytometry profiles and numbers of Aire - Fezf2 - , Aire - Fezf2 + and Aire + Fezf2 + cells in total mTECs, mTEC lo and mTEC hi (F) and of DCKL1 + cells in Aire - mTECs (G) from RipmOVAxOTII- Rag2 -/- and OTII- Rag2 -/- mice. II Abs: secondary antibodies. Data are representative of 2 independent experiments (n=3-4 mice per group and experiment). Error bars show mean◻±◻SEM, *p

    Techniques Used: Flow Cytometry, Hi-C, Mouse Assay, Staining, Derivative Assay

    mTEC lo subset composition is altered in ΔCD4 and mTEC ΔMHCII mice. (A) Confocal images of thymic sections from WT, ΔCD4 and mTEC ΔMHCII mice stained for Aire (green) and Fezf2 (red). 12 and 20 sections derived from 2 WT, 2 ΔCD4 and 2 mTEC ΔMHCII mice were quantified. Scale bar, 50 μm. Unfilled, dashed and solid arrowheads indicate Aire + Fezf2 - , Aire - Fezf2 + and Aire + Fezf2 + cells, respectively. The histogram shows the density of Aire + Fezf2 - , Aire - Fezf2 + and Aire + Fezf2 + cells. (B,C) Flow cytometry profiles and numbers of Aire - Fezf2 - , Aire - Fezf2 + and Aire + Fezf2 + cells in total mTECs, mTEC lo and mTEC hi (B) and of DCKL1 + cells in Aire - mTECs (C) from WT, ΔCD4 and mTEC ΔMHCII mice. II Abs: secondary antibodies. Data are representative of 2-3 independent experiments (n=3-4 mice per group and experiment). Error bars show mean◻±◻SEM, *p
    Figure Legend Snippet: mTEC lo subset composition is altered in ΔCD4 and mTEC ΔMHCII mice. (A) Confocal images of thymic sections from WT, ΔCD4 and mTEC ΔMHCII mice stained for Aire (green) and Fezf2 (red). 12 and 20 sections derived from 2 WT, 2 ΔCD4 and 2 mTEC ΔMHCII mice were quantified. Scale bar, 50 μm. Unfilled, dashed and solid arrowheads indicate Aire + Fezf2 - , Aire - Fezf2 + and Aire + Fezf2 + cells, respectively. The histogram shows the density of Aire + Fezf2 - , Aire - Fezf2 + and Aire + Fezf2 + cells. (B,C) Flow cytometry profiles and numbers of Aire - Fezf2 - , Aire - Fezf2 + and Aire + Fezf2 + cells in total mTECs, mTEC lo and mTEC hi (B) and of DCKL1 + cells in Aire - mTECs (C) from WT, ΔCD4 and mTEC ΔMHCII mice. II Abs: secondary antibodies. Data are representative of 2-3 independent experiments (n=3-4 mice per group and experiment). Error bars show mean◻±◻SEM, *p

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

    10) Product Images from "Structure of a patient-derived antibody in complex with allergen reveals simultaneous conventional and superantigen-like recognition"

    Article Title: Structure of a patient-derived antibody in complex with allergen reveals simultaneous conventional and superantigen-like recognition

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1806840115

    Characterization of oligomeric state and basophil degranulation for the three polcalcin allergens. ( A ) Chromatograms of the RI signals for Phl p 7, Bet v 4, and Ole e 3. The three proteins [in 25 mM Tris⋅HCl (pH 7.4) and 150 mM NaCl with 4 mM CaCl 2 ] were run over a Superdex 75 5/150 column (GE Healthcare) at a flow rate of 0.3 mL/min. The differential RI is shown in black (A.U., arbitrary units). The green lines across the protein elution volumes show the molecular masses of the proteins, indicating a monomeric state. ( B ) Basophil degranulation assay showing the percentage degranulation of RBL-SX38 cells sensitized with 102.1F10 IgE (black bars) and CS09G6K IgE (white bars) antibodies followed by stimulation with 5,000 ng/μL of Phl p 7, Bet v 4, or Ole e 3 or with assay medium (background). Degranulation is expressed as a percentage of that induced by cell lysis. Bars represent the mean ± SEM of three independent experiments. Statistical significance was determined by t test; **** P
    Figure Legend Snippet: Characterization of oligomeric state and basophil degranulation for the three polcalcin allergens. ( A ) Chromatograms of the RI signals for Phl p 7, Bet v 4, and Ole e 3. The three proteins [in 25 mM Tris⋅HCl (pH 7.4) and 150 mM NaCl with 4 mM CaCl 2 ] were run over a Superdex 75 5/150 column (GE Healthcare) at a flow rate of 0.3 mL/min. The differential RI is shown in black (A.U., arbitrary units). The green lines across the protein elution volumes show the molecular masses of the proteins, indicating a monomeric state. ( B ) Basophil degranulation assay showing the percentage degranulation of RBL-SX38 cells sensitized with 102.1F10 IgE (black bars) and CS09G6K IgE (white bars) antibodies followed by stimulation with 5,000 ng/μL of Phl p 7, Bet v 4, or Ole e 3 or with assay medium (background). Degranulation is expressed as a percentage of that induced by cell lysis. Bars represent the mean ± SEM of three independent experiments. Statistical significance was determined by t test; **** P

    Techniques Used: Flow Cytometry, Degranulation Assay, Lysis

    11) Product Images from "Dickkopf-3 links HSF1 and YAP/TAZ signalling to control aggressive behaviours in cancer-associated fibroblasts"

    Article Title: Dickkopf-3 links HSF1 and YAP/TAZ signalling to control aggressive behaviours in cancer-associated fibroblasts

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07987-0

    DKK3 is upregulated in the stroma of breast, colon and ovarian cancers. a Tukey boxplots showing z-score values of DKK3 mRNA expression in normal and cancerous stroma from breast, colorectal and ovarian cancers (Breast: normal, n = 6; cancer, n = 53. Colon: normal, n = 4; cancer, n = 13. Ovary: normal, n = 8; cancer, n = 31). b Representative images of DKK3 staining in breast, colorectal and ovarian cancers and normal tissues. Scale bar, 100 µm. c Tukey boxplots showing quantification of DKK3 staining (Histoscore) in breast, colorectal and ovarian cancers and normal tissue counterparts (Breast: normal/adjacent, n = 9; cancer, n = 109. Colon: normal/adjacent, n = 14; cancer, n = 107. Ovary: normal/adjacent, n = 8; cancer, n = 138). d Tukey boxplots showing DKK3 Histoscore in non-invasive breast cancers (Stage 1 2), invasive breast-cancers (Stage 3 4) and normal tissue counterparts. Left graph shows all cancers irrespective of their subtype (normal, n = 9; Stage 1 2, n = 74; Stage 3 4, n = 74). Middle graph shows ER-negative breast cancers (normal, n = 9; Stage 1 2, n = 40; Stage 3 4, n = 40). Right graphs shows ER-positive breast cancers (normal, n = 9; Stage 1 2, n = 21; Stage 3 4, n = 21). e Disease-free survival of breast cancer patients stratified on stromal DKK3 gene expression (GSE9014, ER-negative patients). f Images show DKK3 (green), vimentin (VIM; red) and DAPI (blue) staining of two representative human breast cancer tissues. Scale bar, 50 µm. g Tukey boxplot shows Dkk3 mRNA expression levels (relative to Gapdh ) in Cancer cells (Epcam + ), immune cells (Cd45 + ), endothelial cells (Cd31 + ) and fibroblasts (Pdgfra + ) from MMTV-PyMT tumorus ( n = 4). h Graphs show correlations between the expression of DKK3 and ACTA2 , FAP and COL1A2 in normal and cancerous stroma from mammary gland (GSE9014), colorectal (GSE35602) and ovarian (GSE40595) human tissues. Pearson correlation coefficient (r) is shown. Each dot represents z-score values from individual patients. i Kaplan–Meier curves of recurrence-free survival, disease-specific survival and progression-free survival of ER-negative breast cancer, colorectal cancer and ovarian cancer patients, respectively, based on DKK3 and DKK2 gene expression. Where indicated, individual p values are shown; alternatively the following symbols were used to describe statistical significance: * P
    Figure Legend Snippet: DKK3 is upregulated in the stroma of breast, colon and ovarian cancers. a Tukey boxplots showing z-score values of DKK3 mRNA expression in normal and cancerous stroma from breast, colorectal and ovarian cancers (Breast: normal, n = 6; cancer, n = 53. Colon: normal, n = 4; cancer, n = 13. Ovary: normal, n = 8; cancer, n = 31). b Representative images of DKK3 staining in breast, colorectal and ovarian cancers and normal tissues. Scale bar, 100 µm. c Tukey boxplots showing quantification of DKK3 staining (Histoscore) in breast, colorectal and ovarian cancers and normal tissue counterparts (Breast: normal/adjacent, n = 9; cancer, n = 109. Colon: normal/adjacent, n = 14; cancer, n = 107. Ovary: normal/adjacent, n = 8; cancer, n = 138). d Tukey boxplots showing DKK3 Histoscore in non-invasive breast cancers (Stage 1 2), invasive breast-cancers (Stage 3 4) and normal tissue counterparts. Left graph shows all cancers irrespective of their subtype (normal, n = 9; Stage 1 2, n = 74; Stage 3 4, n = 74). Middle graph shows ER-negative breast cancers (normal, n = 9; Stage 1 2, n = 40; Stage 3 4, n = 40). Right graphs shows ER-positive breast cancers (normal, n = 9; Stage 1 2, n = 21; Stage 3 4, n = 21). e Disease-free survival of breast cancer patients stratified on stromal DKK3 gene expression (GSE9014, ER-negative patients). f Images show DKK3 (green), vimentin (VIM; red) and DAPI (blue) staining of two representative human breast cancer tissues. Scale bar, 50 µm. g Tukey boxplot shows Dkk3 mRNA expression levels (relative to Gapdh ) in Cancer cells (Epcam + ), immune cells (Cd45 + ), endothelial cells (Cd31 + ) and fibroblasts (Pdgfra + ) from MMTV-PyMT tumorus ( n = 4). h Graphs show correlations between the expression of DKK3 and ACTA2 , FAP and COL1A2 in normal and cancerous stroma from mammary gland (GSE9014), colorectal (GSE35602) and ovarian (GSE40595) human tissues. Pearson correlation coefficient (r) is shown. Each dot represents z-score values from individual patients. i Kaplan–Meier curves of recurrence-free survival, disease-specific survival and progression-free survival of ER-negative breast cancer, colorectal cancer and ovarian cancer patients, respectively, based on DKK3 and DKK2 gene expression. Where indicated, individual p values are shown; alternatively the following symbols were used to describe statistical significance: * P

    Techniques Used: Expressing, Staining

    12) Product Images from "Lack of FcRn Impairs Natural Killer Cell Development and Functions in the Tumor Microenvironment"

    Article Title: Lack of FcRn Impairs Natural Killer Cell Development and Functions in the Tumor Microenvironment

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02259

    Influence of FcRn on NK cell functions and in vitro expansion. Purified splenic NK cells were analyzed by flow cytometry for (A) the intracellular measurement of IFN-γ and (B) the surface expression of the late endosomal marker CD107a, after 4-h incubation at 37°C without (Ctr) or with PMA (100 ng/mL)/ionomycine (500 ng/mL). Data are median ± Min to Max analyzed from eight independent experiments using pooled NK cells from 2 mice. Freshly isolated splenocytes were seeded in RMPI 1640 complete medium supplemented with 5,000 U/ml rhIL2 or with 5 ng/ml rhIL12, 50 ng/ml rhIL15, and 10 ng/ml rhIL18 for 4-h (C,D) . Within splenocytes, CD3 − /NK1.1 + /NKp46 + cells were analyzed for (C) the intracellular expression of IFN-γ and (D) the surface expression of CD107a by flow cytometry. Data are median ± Min to Max from two independent experiments using pooled spleens from 2 mice. (E) Cytotoxicity assay was performed against CFSE-labeled YAC-1 target cells with different ratios of purified NK cells previously maintained overnight in RPMI 1640 complete medium supplemented with 50 U/ml of rhIL2 ( n = 3). The results were expressed as means ± SEM. (F,G) Purified splenic NK cells were plated in complete medium supplemented with 5,000 U/ml rhIL2. (F) The living cell numbers and (G) the percentage of dead cells were determined daily by manual cell counting using trypan blue in Malassez chamber ( n = 3). The results were expressed as mean ± SEM (F) and median ± Min to Max (G) . ns = not significant * p ≤ 0.05 and ** p ≤ 0.005 using two-tailed non-parametric and unpaired Wilcoxon-Mann-Whitney test.
    Figure Legend Snippet: Influence of FcRn on NK cell functions and in vitro expansion. Purified splenic NK cells were analyzed by flow cytometry for (A) the intracellular measurement of IFN-γ and (B) the surface expression of the late endosomal marker CD107a, after 4-h incubation at 37°C without (Ctr) or with PMA (100 ng/mL)/ionomycine (500 ng/mL). Data are median ± Min to Max analyzed from eight independent experiments using pooled NK cells from 2 mice. Freshly isolated splenocytes were seeded in RMPI 1640 complete medium supplemented with 5,000 U/ml rhIL2 or with 5 ng/ml rhIL12, 50 ng/ml rhIL15, and 10 ng/ml rhIL18 for 4-h (C,D) . Within splenocytes, CD3 − /NK1.1 + /NKp46 + cells were analyzed for (C) the intracellular expression of IFN-γ and (D) the surface expression of CD107a by flow cytometry. Data are median ± Min to Max from two independent experiments using pooled spleens from 2 mice. (E) Cytotoxicity assay was performed against CFSE-labeled YAC-1 target cells with different ratios of purified NK cells previously maintained overnight in RPMI 1640 complete medium supplemented with 50 U/ml of rhIL2 ( n = 3). The results were expressed as means ± SEM. (F,G) Purified splenic NK cells were plated in complete medium supplemented with 5,000 U/ml rhIL2. (F) The living cell numbers and (G) the percentage of dead cells were determined daily by manual cell counting using trypan blue in Malassez chamber ( n = 3). The results were expressed as mean ± SEM (F) and median ± Min to Max (G) . ns = not significant * p ≤ 0.05 and ** p ≤ 0.005 using two-tailed non-parametric and unpaired Wilcoxon-Mann-Whitney test.

    Techniques Used: In Vitro, Purification, Flow Cytometry, Cytometry, Expressing, Marker, Incubation, Mouse Assay, Isolation, Cytotoxicity Assay, Labeling, Cell Counting, Two Tailed Test, MANN-WHITNEY

    13) Product Images from "Adoptive Transfer of Immunomodulatory M2 Macrophages Prevents Type 1 Diabetes in NOD Mice"

    Article Title: Adoptive Transfer of Immunomodulatory M2 Macrophages Prevents Type 1 Diabetes in NOD Mice

    Journal: Diabetes

    doi: 10.2337/db11-1635

    M2r macrophages suppress ex vivo T-cell activity. M2r or M0 macrophages (2.5 × 10 6 ) were intraperitoneally injected into 12–16-week-old NOD-BDC2.5 mice. A : PLNs were dissected after 1 week, and lymphocytes were restimulated with BDC2.5 mimotope for 72 h to induce proliferation. Readout was CPM . B : PLNs were dissected after 1 week, and lymphocyte subsets were analyzed by flow cytometry for cell numbers (percent and absolute values). C : Activation status of CD4 + subset assessed by CD44 and CD62L expression. D : Pancreata were dissected after 1 week, and lymphocyte subsets were analyzed by flow cytometry for cell numbers (percent and absolute values). E : M2r macrophages (2–3 × 10 6 ) or control (PBS) were intraperitoneally injected into 12–13-week-old NOD-FoxP3-GFP mice, PLNs were dissected after 1 week, and T-cell subset numbers were analyzed. The data from A – D represent two independent experiments ( n = 4). The data in E represent pooled data from four independent experiments. Error bars are presented in SEM. * P
    Figure Legend Snippet: M2r macrophages suppress ex vivo T-cell activity. M2r or M0 macrophages (2.5 × 10 6 ) were intraperitoneally injected into 12–16-week-old NOD-BDC2.5 mice. A : PLNs were dissected after 1 week, and lymphocytes were restimulated with BDC2.5 mimotope for 72 h to induce proliferation. Readout was CPM . B : PLNs were dissected after 1 week, and lymphocyte subsets were analyzed by flow cytometry for cell numbers (percent and absolute values). C : Activation status of CD4 + subset assessed by CD44 and CD62L expression. D : Pancreata were dissected after 1 week, and lymphocyte subsets were analyzed by flow cytometry for cell numbers (percent and absolute values). E : M2r macrophages (2–3 × 10 6 ) or control (PBS) were intraperitoneally injected into 12–13-week-old NOD-FoxP3-GFP mice, PLNs were dissected after 1 week, and T-cell subset numbers were analyzed. The data from A – D represent two independent experiments ( n = 4). The data in E represent pooled data from four independent experiments. Error bars are presented in SEM. * P

    Techniques Used: Ex Vivo, Activity Assay, Injection, Mouse Assay, Flow Cytometry, Cytometry, Activation Assay, Expressing

    Macrophages predominantly migrate to the pancreas and PLN. A : NOD mice (10–12 weeks of age) received 3 × 10 6 DiR-labeled M2r or M0 macrophages intraperitoneally. Mice were anesthetized prior to anterior imaging after 2 h and 1, 3, 6, and 8 days post–macrophage injection. Bright yellow or dark red represents high or low photon counts, respectively, and white arrows indicate injection sites. Two individual mice were analyzed, and the data represent two individual experiments. B : After transfer, the liver, kidney, spleen, pancreas, and PLNs were dissected after 3 days post–macrophage injection. The image is representative of eight individual mice (four treated with M0 and four with M2r). C : Photon count from each organ in B was quantified, and control organ photon count was subtracted to obtain the real macrophage emission. Error bars are presented in SEM. D : NOD mice received 2 × 10 6 DiI-labeled M2r macrophages (red) intraperitoneally, and pancreata were dissected at day 3 and stained for CD11b (green) and DAPI (blue). The data are representative of four different mice. (A high-quality digital representation of this figure is available in the online issue.)
    Figure Legend Snippet: Macrophages predominantly migrate to the pancreas and PLN. A : NOD mice (10–12 weeks of age) received 3 × 10 6 DiR-labeled M2r or M0 macrophages intraperitoneally. Mice were anesthetized prior to anterior imaging after 2 h and 1, 3, 6, and 8 days post–macrophage injection. Bright yellow or dark red represents high or low photon counts, respectively, and white arrows indicate injection sites. Two individual mice were analyzed, and the data represent two individual experiments. B : After transfer, the liver, kidney, spleen, pancreas, and PLNs were dissected after 3 days post–macrophage injection. The image is representative of eight individual mice (four treated with M0 and four with M2r). C : Photon count from each organ in B was quantified, and control organ photon count was subtracted to obtain the real macrophage emission. Error bars are presented in SEM. D : NOD mice received 2 × 10 6 DiI-labeled M2r macrophages (red) intraperitoneally, and pancreata were dissected at day 3 and stained for CD11b (green) and DAPI (blue). The data are representative of four different mice. (A high-quality digital representation of this figure is available in the online issue.)

    Techniques Used: Mouse Assay, Labeling, Imaging, Injection, Staining

    M2r macrophages retain an M2 signature after secondary proinflammatory stimulation both in vitro and in vivo. A : Schematic experimental set-up. Primary activation with M0-, M2r-, or M1-inducing stimuli was for 24 h, and secondary activation for an additional 24 h with LPS/IFNγ (48 h). B : IL-10 production assessed by ELISA in secondary-activated macrophages. C : Flow cytometry analyses of CD86 and PD-L2 expression on primary- and secondary-activated macrophages. Histograms and mean fluorescence intensity analyses are depicted. MFI, mean fluorescence intensity. D : Selected gene expression assessed by RT-PCR after primary and secondary activations of macrophages. E : Ex vivo analyses of CD86 and PD-L2 on CD11b + DiD + macrophages recovered from pancreata ( left ) or PLNs ( right ). Histograms and percentage of cells depicted. The results are representative of two independent experiments ( n = 4). * P
    Figure Legend Snippet: M2r macrophages retain an M2 signature after secondary proinflammatory stimulation both in vitro and in vivo. A : Schematic experimental set-up. Primary activation with M0-, M2r-, or M1-inducing stimuli was for 24 h, and secondary activation for an additional 24 h with LPS/IFNγ (48 h). B : IL-10 production assessed by ELISA in secondary-activated macrophages. C : Flow cytometry analyses of CD86 and PD-L2 expression on primary- and secondary-activated macrophages. Histograms and mean fluorescence intensity analyses are depicted. MFI, mean fluorescence intensity. D : Selected gene expression assessed by RT-PCR after primary and secondary activations of macrophages. E : Ex vivo analyses of CD86 and PD-L2 on CD11b + DiD + macrophages recovered from pancreata ( left ) or PLNs ( right ). Histograms and percentage of cells depicted. The results are representative of two independent experiments ( n = 4). * P

    Techniques Used: In Vitro, In Vivo, Activation Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Expressing, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Ex Vivo

    M2r macrophages protect NOD mice from T1D by protecting pancreatic β-cells. A : IL-4/IL-10/TGF-β–stimulated macrophages (2.5 × 10 6 ) (M2r, blue, n = 12), untreated macrophages (M0, red, n = 8), or vehicle (PBS, black, n = 8) were intraperitoneally injected into 16-week-old prediabetic NOD mice (arrow). The M2r-treated group was significantly protected compared with M0 and PBS groups, as independently statistically analyzed using the Mantel-Cox test in a Kaplan-Meier survival graph. These results are representative data from two independent experiments with a similar outcome. B : Organs were stained with anti-insulin (red) and anti-CD3 (green)–specific antibodies prior to three-dimensional reconstruction using optical projection tomography. Yellow represents colocalization of CD3 and insulin staining, indicating insulitis. Organs from 16-week-old mice represent three individual animals; 21- and 26-week-old organs represent two individual animals. C : After M2r or M0 macrophage transfer, pancreata were dissected 8 weeks later (24 weeks of age), and cryosections were stained with anti-insulin (red) and anti-CD3 (green) ( n = 5). D : Insulin + islets were counted manually from the sections in two to five transverse sections per animal to obtain the average number of insulin + islets per section ( n = 4). * P
    Figure Legend Snippet: M2r macrophages protect NOD mice from T1D by protecting pancreatic β-cells. A : IL-4/IL-10/TGF-β–stimulated macrophages (2.5 × 10 6 ) (M2r, blue, n = 12), untreated macrophages (M0, red, n = 8), or vehicle (PBS, black, n = 8) were intraperitoneally injected into 16-week-old prediabetic NOD mice (arrow). The M2r-treated group was significantly protected compared with M0 and PBS groups, as independently statistically analyzed using the Mantel-Cox test in a Kaplan-Meier survival graph. These results are representative data from two independent experiments with a similar outcome. B : Organs were stained with anti-insulin (red) and anti-CD3 (green)–specific antibodies prior to three-dimensional reconstruction using optical projection tomography. Yellow represents colocalization of CD3 and insulin staining, indicating insulitis. Organs from 16-week-old mice represent three individual animals; 21- and 26-week-old organs represent two individual animals. C : After M2r or M0 macrophage transfer, pancreata were dissected 8 weeks later (24 weeks of age), and cryosections were stained with anti-insulin (red) and anti-CD3 (green) ( n = 5). D : Insulin + islets were counted manually from the sections in two to five transverse sections per animal to obtain the average number of insulin + islets per section ( n = 4). * P

    Techniques Used: Mouse Assay, Injection, Staining

    14) Product Images from "Lack of FcRn Impairs Natural Killer Cell Development and Functions in the Tumor Microenvironment"

    Article Title: Lack of FcRn Impairs Natural Killer Cell Development and Functions in the Tumor Microenvironment

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02259

    Influence of FcRn on NK cell functions and in vitro expansion. Purified splenic NK cells were analyzed by flow cytometry for (A) the intracellular measurement of IFN-γ and (B) the surface expression of the late endosomal marker CD107a, after 4-h incubation at 37°C without (Ctr) or with PMA (100 ng/mL)/ionomycine (500 ng/mL). Data are median ± Min to Max analyzed from eight independent experiments using pooled NK cells from 2 mice. Freshly isolated splenocytes were seeded in RMPI 1640 complete medium supplemented with 5,000 U/ml rhIL2 or with 5 ng/ml rhIL12, 50 ng/ml rhIL15, and 10 ng/ml rhIL18 for 4-h (C,D) . Within splenocytes, CD3 − /NK1.1 + /NKp46 + cells were analyzed for (C) the intracellular expression of IFN-γ and (D) the surface expression of CD107a by flow cytometry. Data are median ± Min to Max from two independent experiments using pooled spleens from 2 mice. (E) Cytotoxicity assay was performed against CFSE-labeled YAC-1 target cells with different ratios of purified NK cells previously maintained overnight in RPMI 1640 complete medium supplemented with 50 U/ml of rhIL2 ( n = 3). The results were expressed as means ± SEM. (F,G) Purified splenic NK cells were plated in complete medium supplemented with 5,000 U/ml rhIL2. (F) The living cell numbers and (G) the percentage of dead cells were determined daily by manual cell counting using trypan blue in Malassez chamber ( n = 3). The results were expressed as mean ± SEM (F) and median ± Min to Max (G) . ns = not significant * p ≤ 0.05 and ** p ≤ 0.005 using two-tailed non-parametric and unpaired Wilcoxon-Mann-Whitney test.
    Figure Legend Snippet: Influence of FcRn on NK cell functions and in vitro expansion. Purified splenic NK cells were analyzed by flow cytometry for (A) the intracellular measurement of IFN-γ and (B) the surface expression of the late endosomal marker CD107a, after 4-h incubation at 37°C without (Ctr) or with PMA (100 ng/mL)/ionomycine (500 ng/mL). Data are median ± Min to Max analyzed from eight independent experiments using pooled NK cells from 2 mice. Freshly isolated splenocytes were seeded in RMPI 1640 complete medium supplemented with 5,000 U/ml rhIL2 or with 5 ng/ml rhIL12, 50 ng/ml rhIL15, and 10 ng/ml rhIL18 for 4-h (C,D) . Within splenocytes, CD3 − /NK1.1 + /NKp46 + cells were analyzed for (C) the intracellular expression of IFN-γ and (D) the surface expression of CD107a by flow cytometry. Data are median ± Min to Max from two independent experiments using pooled spleens from 2 mice. (E) Cytotoxicity assay was performed against CFSE-labeled YAC-1 target cells with different ratios of purified NK cells previously maintained overnight in RPMI 1640 complete medium supplemented with 50 U/ml of rhIL2 ( n = 3). The results were expressed as means ± SEM. (F,G) Purified splenic NK cells were plated in complete medium supplemented with 5,000 U/ml rhIL2. (F) The living cell numbers and (G) the percentage of dead cells were determined daily by manual cell counting using trypan blue in Malassez chamber ( n = 3). The results were expressed as mean ± SEM (F) and median ± Min to Max (G) . ns = not significant * p ≤ 0.05 and ** p ≤ 0.005 using two-tailed non-parametric and unpaired Wilcoxon-Mann-Whitney test.

    Techniques Used: In Vitro, Purification, Flow Cytometry, Cytometry, Expressing, Marker, Incubation, Mouse Assay, Isolation, Cytotoxicity Assay, Labeling, Cell Counting, Two Tailed Test, MANN-WHITNEY

    15) Product Images from "Wheat germ agglutinin–conjugated fluorescent pH sensors for visualizing proton fluxes"

    Article Title: Wheat germ agglutinin–conjugated fluorescent pH sensors for visualizing proton fluxes

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.201912498

    WGA labeling and voltage-clamp fluorometry of CHO cells expressing voltage-gated channels. (A) hHv-1 (top) and Shaker-IR (bottom) currents were recorded before (left) and after (right) WGA labeling (50 µg/ml). Middle panels are currents before (black) and after (red) WGA wash-in at 60 mV. Cells were held at −80 mV, hHv-1 depolarized for 0.5 s from −50 to 100 in 10-mV increments, and Shaker-IR depolarized for 0.2 s from −70 to 60 mV in 10-mV increments. (B) G-V curves of hHv-1 (V 0.5 = 68 ± 6 mV, n = 8, triangles) and Shaker-IR (V 0.5 = −18 ± 1 mV, n = 5, circles) before (closed) and after (open) WGA treatment. hHv-1: V 0.5 = 67 ± 8 mV, n = 9; Shaker-IR: V 0.5 = −11 ± 1 mV, n = 6. (C) Voltage-clamp fluorometry traces from cells labeled with WGA-pHRho (red) and homemade WGA-fluorescein (green). hHv-1 (left) was held at −80 mV, and changes in fluorescence were elicited from 4-s depolarizations from 0 to 100 mV in 20-mV increments. Shaker-IR R362H (right) was held at 30 mV, and fluorescence was elicited from 4-s command voltages from −120 to −20 mV in 20-mV increments. (D) Representative fluorescent images of CHO cells 10–210 min after labeling with WGA-pHRho for 30 min; scale bars represent 10 µm. (E) Bar graph of ΔF/F 0 from hHv-1 expressing cells after a 4-s 80-mV depolarization versus time after WGA-pHRho labeling. n = 3 experiments. Error bars represent SEM.
    Figure Legend Snippet: WGA labeling and voltage-clamp fluorometry of CHO cells expressing voltage-gated channels. (A) hHv-1 (top) and Shaker-IR (bottom) currents were recorded before (left) and after (right) WGA labeling (50 µg/ml). Middle panels are currents before (black) and after (red) WGA wash-in at 60 mV. Cells were held at −80 mV, hHv-1 depolarized for 0.5 s from −50 to 100 in 10-mV increments, and Shaker-IR depolarized for 0.2 s from −70 to 60 mV in 10-mV increments. (B) G-V curves of hHv-1 (V 0.5 = 68 ± 6 mV, n = 8, triangles) and Shaker-IR (V 0.5 = −18 ± 1 mV, n = 5, circles) before (closed) and after (open) WGA treatment. hHv-1: V 0.5 = 67 ± 8 mV, n = 9; Shaker-IR: V 0.5 = −11 ± 1 mV, n = 6. (C) Voltage-clamp fluorometry traces from cells labeled with WGA-pHRho (red) and homemade WGA-fluorescein (green). hHv-1 (left) was held at −80 mV, and changes in fluorescence were elicited from 4-s depolarizations from 0 to 100 mV in 20-mV increments. Shaker-IR R362H (right) was held at 30 mV, and fluorescence was elicited from 4-s command voltages from −120 to −20 mV in 20-mV increments. (D) Representative fluorescent images of CHO cells 10–210 min after labeling with WGA-pHRho for 30 min; scale bars represent 10 µm. (E) Bar graph of ΔF/F 0 from hHv-1 expressing cells after a 4-s 80-mV depolarization versus time after WGA-pHRho labeling. n = 3 experiments. Error bars represent SEM.

    Techniques Used: Whole Genome Amplification, Labeling, Expressing, Fluorescence

    Cell surface pH calibration of WGA-pHRho and WGA-fluorescein. (A) CHO cells were labeled with either WGA-fluorescein or WGA-pHRho, and the change of fluorescence (ΔF/F 0 %) was plotted using pH standards (HEPES; 1 mM, open symbols; 10 mM, closed symbols); F o was chosen to be at the approximate pKa of each sensor (pH 7.0 for WGA-pHRho and pH 6.5 for WGA-fluorescein). The linear fits for WGA-pHRho were pH = 6.99 − 0.06 × [ΔF/F 0 ] and pH = 6.91 − 0.07 × [ΔF/F 0 ] for 1 and 10 mM, respectively; the linear fits for WGA-fluorescein were pH = 6.51 + 0.05 × [ΔF/F0] and pH = 6.52 + 0.05 × [ΔF/F 0 ] for 1 and 10 mM, respectively. (B) Conversion of ΔF/F 0 (%) to CHO cells expressing hHv-1 were held at −80 mV, and changes in fluorescence were elicited from 4-s depolarizations from 0 to 100 mV in 20-mV increments and converted into ΔpH using the calibration curves in A; WGA-pHRho, red; WGA-fluorescein, green. (C) ΔF/F 0 images of cells expressing hHv-1 taken at 4 s shown in B. Top: WGA-pHRho; bottom: WGA-fluorescein. Dotted white circles indicate the voltage-clamped cell. White scale bars represent 10 μm.
    Figure Legend Snippet: Cell surface pH calibration of WGA-pHRho and WGA-fluorescein. (A) CHO cells were labeled with either WGA-fluorescein or WGA-pHRho, and the change of fluorescence (ΔF/F 0 %) was plotted using pH standards (HEPES; 1 mM, open symbols; 10 mM, closed symbols); F o was chosen to be at the approximate pKa of each sensor (pH 7.0 for WGA-pHRho and pH 6.5 for WGA-fluorescein). The linear fits for WGA-pHRho were pH = 6.99 − 0.06 × [ΔF/F 0 ] and pH = 6.91 − 0.07 × [ΔF/F 0 ] for 1 and 10 mM, respectively; the linear fits for WGA-fluorescein were pH = 6.51 + 0.05 × [ΔF/F0] and pH = 6.52 + 0.05 × [ΔF/F 0 ] for 1 and 10 mM, respectively. (B) Conversion of ΔF/F 0 (%) to CHO cells expressing hHv-1 were held at −80 mV, and changes in fluorescence were elicited from 4-s depolarizations from 0 to 100 mV in 20-mV increments and converted into ΔpH using the calibration curves in A; WGA-pHRho, red; WGA-fluorescein, green. (C) ΔF/F 0 images of cells expressing hHv-1 taken at 4 s shown in B. Top: WGA-pHRho; bottom: WGA-fluorescein. Dotted white circles indicate the voltage-clamped cell. White scale bars represent 10 μm.

    Techniques Used: Whole Genome Amplification, Labeling, Fluorescence, Expressing

    Structured light microscopy of lactate-stimulated proton transport in rat ventricular myocytes labeled with WGA-pHRho. (A) A ∼750-nm illuminated z -plane of a WGA-pHRho–labeled ventricular myocyte ∼2 μm above the coverslip focal plane. (B) Time course of 10 mM lactate perfusion before (red) and after addition of 50 nM AR-C155858 (gray). Plotted data are mean + SEM (shading) for clarity ( n = 6 cells); data were collected at 1 Hz. (C) Pseudocolor image depicting the eroding pixel (5 px) analysis to generate the four annuli. (D) Changes of ΔF/F 0 before, during, and after perfusion of 10 mM lactate at each annulus (A0–A3). (E) Pixel intensity histogram of the four annuli (area normalized) for cell shown in A. (F) Average maximal |ΔF/F 0 | upon wash in (solid bars) and wash out (open bars) of 10 mM lactate for nine cells. One-way ANOVA (Bonferroni's multiple comparison test) was used to determine significance (*, P
    Figure Legend Snippet: Structured light microscopy of lactate-stimulated proton transport in rat ventricular myocytes labeled with WGA-pHRho. (A) A ∼750-nm illuminated z -plane of a WGA-pHRho–labeled ventricular myocyte ∼2 μm above the coverslip focal plane. (B) Time course of 10 mM lactate perfusion before (red) and after addition of 50 nM AR-C155858 (gray). Plotted data are mean + SEM (shading) for clarity ( n = 6 cells); data were collected at 1 Hz. (C) Pseudocolor image depicting the eroding pixel (5 px) analysis to generate the four annuli. (D) Changes of ΔF/F 0 before, during, and after perfusion of 10 mM lactate at each annulus (A0–A3). (E) Pixel intensity histogram of the four annuli (area normalized) for cell shown in A. (F) Average maximal |ΔF/F 0 | upon wash in (solid bars) and wash out (open bars) of 10 mM lactate for nine cells. One-way ANOVA (Bonferroni's multiple comparison test) was used to determine significance (*, P

    Techniques Used: Light Microscopy, Labeling, Whole Genome Amplification

    Proton efflux from primary neuron–astrocyte cocultures labeled with WGA-pHRho. (A) Merged Airyscan image of the neuronal and astrocyte planes labeled with WGA-pHRho (red) and DAPI (blue). (B) Pseudocolor image showing the averaged ΔF/F 0 of the regions of interest (ROI) in A after glucose addition. Red shades indicate more activity over time. ROI are denoted by white boxes (A, astrocyte; N, neuron). Data were collected at 2 Hz. Scale bars represent 10 μm in A and B. (C) Magnified images of two astrocytes and neurons in B to show labeling coverage; 8-bit pseudo-color intensity scale bar is for panels in B and C. Scale bars represent 2 μm. (D) Cartoon depiction of the metabolic pathways expected to be affected by glucose starvation and rotenone mitochondrial poisoning. (E and F) Time courses of the (E) neuronal and (F) astrocytic pHRho signal during glucose (3 mM) and rotenone (100 µM) addition for the exemplar in B. (G) Averaged data from E and F (total number of cells in the plot: four neurons and four astrocytes from one single dish; this average is considered n = 1). Error bars represent SEM. (H) Variation of the neuronal and astrocytic responses to glucose and rotenone; 11–12 ROIs from dishes from three animals. (I) Maximal change in ΔF/F 0 ( n = 4–6 dishes) for glucose and rotenone treatment, comprising a total of 19 neurons and 21 astrocytes in total; error bars represent SEM. A paired t test was used to determine significance (*, P
    Figure Legend Snippet: Proton efflux from primary neuron–astrocyte cocultures labeled with WGA-pHRho. (A) Merged Airyscan image of the neuronal and astrocyte planes labeled with WGA-pHRho (red) and DAPI (blue). (B) Pseudocolor image showing the averaged ΔF/F 0 of the regions of interest (ROI) in A after glucose addition. Red shades indicate more activity over time. ROI are denoted by white boxes (A, astrocyte; N, neuron). Data were collected at 2 Hz. Scale bars represent 10 μm in A and B. (C) Magnified images of two astrocytes and neurons in B to show labeling coverage; 8-bit pseudo-color intensity scale bar is for panels in B and C. Scale bars represent 2 μm. (D) Cartoon depiction of the metabolic pathways expected to be affected by glucose starvation and rotenone mitochondrial poisoning. (E and F) Time courses of the (E) neuronal and (F) astrocytic pHRho signal during glucose (3 mM) and rotenone (100 µM) addition for the exemplar in B. (G) Averaged data from E and F (total number of cells in the plot: four neurons and four astrocytes from one single dish; this average is considered n = 1). Error bars represent SEM. (H) Variation of the neuronal and astrocytic responses to glucose and rotenone; 11–12 ROIs from dishes from three animals. (I) Maximal change in ΔF/F 0 ( n = 4–6 dishes) for glucose and rotenone treatment, comprising a total of 19 neurons and 21 astrocytes in total; error bars represent SEM. A paired t test was used to determine significance (*, P

    Techniques Used: Labeling, Whole Genome Amplification, Activity Assay

    16) Product Images from "Subventricular zone adult mouse neural stem cells and human glioblastoma stem cells require insulin receptor for self-renewal"

    Article Title: Subventricular zone adult mouse neural stem cells and human glioblastoma stem cells require insulin receptor for self-renewal

    Journal: bioRxiv

    doi: 10.1101/2020.03.10.985598

    IR knockout decreases NSC population. Neurospheres generated from ΔNSC-IR WT and ΔNSC-IR KO mice were treated with 0.5 µM of 4-OH tamoxifen or vehicle for 24 hour followed by dissociation for flow cytometry. A . Control groups showing similar CD133, LeX, NG2 and CD140a expression. B. NSC numbers in ΔNSC-IR WT (control) induced with tamoxifen C. NSC numbers in ΔNSC-IR KO induced with tamoxifen. Representative data of one flow experiment.
    Figure Legend Snippet: IR knockout decreases NSC population. Neurospheres generated from ΔNSC-IR WT and ΔNSC-IR KO mice were treated with 0.5 µM of 4-OH tamoxifen or vehicle for 24 hour followed by dissociation for flow cytometry. A . Control groups showing similar CD133, LeX, NG2 and CD140a expression. B. NSC numbers in ΔNSC-IR WT (control) induced with tamoxifen C. NSC numbers in ΔNSC-IR KO induced with tamoxifen. Representative data of one flow experiment.

    Techniques Used: Knock-Out, Generated, Mouse Assay, Flow Cytometry, Expressing

    17) Product Images from "Haploinsufficient Rock1+/− and Rock2+/− Mice Are Not Protected from Cardiac Inflammation and Postinflammatory Fibrosis in Experimental Autoimmune Myocarditis"

    Article Title: Haploinsufficient Rock1+/− and Rock2+/− Mice Are Not Protected from Cardiac Inflammation and Postinflammatory Fibrosis in Experimental Autoimmune Myocarditis

    Journal: Cells

    doi: 10.3390/cells9030700

    TGF-β converts cardiac fibroblasts and inflammatory myeloid cells into myofibroblasts. Panel ( A ) shows representative flow cytometry analysis of the indicated antigens for inflammatory myeloid cells isolated from wild-type or Rock1 +/− hearts at day 18-21 of EAM and expanded in vitro. Panel ( B ) shows immunoblots and panel ( C ) the respective quantifications of ROCK1 protein levels (normalized to GAPDH) in wild-type and Rock1 +/− cardiac fibroblasts and inflammatory myeloid cells. Panels ( D ) and ( E ) show representative immunofluorescence for αSMA (red) and F-actin (phalloidin staining, green) of cardiac fibroblasts ( A ) and inflammatory myeloid cells ( B ) cultured in the absence (ctrl) or presence of TGF-β (+TGF-β) for 3 days. Bars represent mean value ± SEM. n = 4, p -value was calculated with the unpaired Student’s t -test.
    Figure Legend Snippet: TGF-β converts cardiac fibroblasts and inflammatory myeloid cells into myofibroblasts. Panel ( A ) shows representative flow cytometry analysis of the indicated antigens for inflammatory myeloid cells isolated from wild-type or Rock1 +/− hearts at day 18-21 of EAM and expanded in vitro. Panel ( B ) shows immunoblots and panel ( C ) the respective quantifications of ROCK1 protein levels (normalized to GAPDH) in wild-type and Rock1 +/− cardiac fibroblasts and inflammatory myeloid cells. Panels ( D ) and ( E ) show representative immunofluorescence for αSMA (red) and F-actin (phalloidin staining, green) of cardiac fibroblasts ( A ) and inflammatory myeloid cells ( B ) cultured in the absence (ctrl) or presence of TGF-β (+TGF-β) for 3 days. Bars represent mean value ± SEM. n = 4, p -value was calculated with the unpaired Student’s t -test.

    Techniques Used: Flow Cytometry, Isolation, In Vitro, Western Blot, Immunofluorescence, Staining, Cell Culture

    Flow cytometry analysis of cardiac inflammatory cells in wild-type and Rock1 +/− mice at the acute stage of myocarditis. Panel ( A ) shows gating strategy for myeloid (CD11b + ) cell subsets and cardiac fibroblasts (EGFP + CD11b – ) in flow cytometry analysis of hearts obtained from mice with the Coll-EGFP reporter transgene. Analysis of unstained heart sample is shown in Supplementary Figure S2 . Quantifications of total number of myeloid and cardiac fibroblasts, as well as indicated myeloid cell subsets in hearts of Coll-EGFP (n = 8) and Rock1 +/− x Coll-EGFP (n = 8) mice, at day 21 of EAM are shown in panels ( B ) and ( C ), respectively. Each dot represents data for one heart, and bars present mean value ± SEM. p -value was calculated with the unpaired Student’s t -test.
    Figure Legend Snippet: Flow cytometry analysis of cardiac inflammatory cells in wild-type and Rock1 +/− mice at the acute stage of myocarditis. Panel ( A ) shows gating strategy for myeloid (CD11b + ) cell subsets and cardiac fibroblasts (EGFP + CD11b – ) in flow cytometry analysis of hearts obtained from mice with the Coll-EGFP reporter transgene. Analysis of unstained heart sample is shown in Supplementary Figure S2 . Quantifications of total number of myeloid and cardiac fibroblasts, as well as indicated myeloid cell subsets in hearts of Coll-EGFP (n = 8) and Rock1 +/− x Coll-EGFP (n = 8) mice, at day 21 of EAM are shown in panels ( B ) and ( C ), respectively. Each dot represents data for one heart, and bars present mean value ± SEM. p -value was calculated with the unpaired Student’s t -test.

    Techniques Used: Flow Cytometry, Mouse Assay

    18) Product Images from "Thy-1 Deficiency Augments Bone Loss in Obesity by Affecting Bone Formation and Resorption"

    Article Title: Thy-1 Deficiency Augments Bone Loss in Obesity by Affecting Bone Formation and Resorption

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2018.00127

    Lack of Thy-1 reduces osteoblast differentiation as well as bone formation and increases osteoclast differentiation and bone resorption in obese mice. Wildtype (WT) and Thy-1 −/− (KO) mice were fed with a high fat diet for 18 weeks (HFD). (A–J) Bone formation and (K-O) bone resorption were analyzed. The (A) osteoblast number per bone perimeter (Ob.N/B.Pm), (B) osteoblast surface per bone surface (Ob.S/BS) were analyzed using histology methods. Gene expression of the osteogenic markers (C) runt-related transcription factor 2 ( Runx2 ), (D) and alkaline phosphatase ( Tnalp ) was analyzed by RT-PCR technique. (E) The serum concentration of total procollagen type 1 amino-terminal propeptide (P1NP) was analyzed using ELISA technique and the (F) osteoid surface per bone perimeter (Osteid.S/B.Pm) by histology. (G) Representative sections of von Kossa/van Gieson staining of bone (black) and cartilage (dense red area close to the bone). (H) The bone formation rate per bone surface (BFR/BS) as well as (I) mineral apposition rate (MAR) were determined by histomorphometric analysis (double calcein labeling). (J) Representative images of calcein labeling (green). (K) Osteoclast number per bone perimeter (Oc.N/B.Pm) and (L) osteoclast surface per bone surface (Oc.S/BS) were analyzed by staining of tartrate resistant acid phosphatase (TRAP). (M) Representative images of TRAP staining (red spots and black arrows = TRAP-positive cell/osteoclast). (N) Serum concentration of the bone resorption marker carboxy-terminal collagen crosslinks (CTX) was measured by ELISA technique. (O) Gene expression of Trap was evaluated via RT-PCR. Each point represents one mouse and median ± SD is presented. Asterisks denote significance level of ∗ P
    Figure Legend Snippet: Lack of Thy-1 reduces osteoblast differentiation as well as bone formation and increases osteoclast differentiation and bone resorption in obese mice. Wildtype (WT) and Thy-1 −/− (KO) mice were fed with a high fat diet for 18 weeks (HFD). (A–J) Bone formation and (K-O) bone resorption were analyzed. The (A) osteoblast number per bone perimeter (Ob.N/B.Pm), (B) osteoblast surface per bone surface (Ob.S/BS) were analyzed using histology methods. Gene expression of the osteogenic markers (C) runt-related transcription factor 2 ( Runx2 ), (D) and alkaline phosphatase ( Tnalp ) was analyzed by RT-PCR technique. (E) The serum concentration of total procollagen type 1 amino-terminal propeptide (P1NP) was analyzed using ELISA technique and the (F) osteoid surface per bone perimeter (Osteid.S/B.Pm) by histology. (G) Representative sections of von Kossa/van Gieson staining of bone (black) and cartilage (dense red area close to the bone). (H) The bone formation rate per bone surface (BFR/BS) as well as (I) mineral apposition rate (MAR) were determined by histomorphometric analysis (double calcein labeling). (J) Representative images of calcein labeling (green). (K) Osteoclast number per bone perimeter (Oc.N/B.Pm) and (L) osteoclast surface per bone surface (Oc.S/BS) were analyzed by staining of tartrate resistant acid phosphatase (TRAP). (M) Representative images of TRAP staining (red spots and black arrows = TRAP-positive cell/osteoclast). (N) Serum concentration of the bone resorption marker carboxy-terminal collagen crosslinks (CTX) was measured by ELISA technique. (O) Gene expression of Trap was evaluated via RT-PCR. Each point represents one mouse and median ± SD is presented. Asterisks denote significance level of ∗ P

    Techniques Used: Mouse Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Labeling, Marker

    Obese Thy-1 −/− mice display decreased trabecular bone mass while cortical bone mass and biomechanical properties are unaltered. Wildtype (WT) and Thy-1 −/− (KO) mice were fed with a high fat diet for 18 weeks (HFD). As control group, WT mice were fed a standard chow (CHOW) for the same time period. (A) The body weight of standard and HFD fed WT and KO mice over 18 weeks. Hashtags denote significance level of # P
    Figure Legend Snippet: Obese Thy-1 −/− mice display decreased trabecular bone mass while cortical bone mass and biomechanical properties are unaltered. Wildtype (WT) and Thy-1 −/− (KO) mice were fed with a high fat diet for 18 weeks (HFD). As control group, WT mice were fed a standard chow (CHOW) for the same time period. (A) The body weight of standard and HFD fed WT and KO mice over 18 weeks. Hashtags denote significance level of # P

    Techniques Used: Mouse Assay

    Lack of Thy-1 promotes obesity mediated inflammation. Wildtype (WT) and Thy-1 −/− (KO) mice were fed with a high fat diet for 18 weeks (HFD). (A) Number of adipocytes per total area (N.Adipo/Tt.Ar) and (B) adipocyte area (Adipo.Ar) of adipocytes were analyzed by histology technique. (C) Gene expression of the fat marker fatty-acid-binding protein ( Fabp ) in bone was assessed by RT-PCR. (D) Fat volume (FV/TV) in the femoral medullary cavity was analyzed by osmium tetroxide staining. (E) Representative 3D-images of the fat volume of whole femur. (F,G) Gene expression of the pro-inflammatory markers tumor necrosis factor α ( Tnfα ) and interleukin 6 ( Il6 ). (H) Osteoclast precursor cells from WT mice were cultured ex vivo without (w/o) and with TNFα and osteoclastogenesis was detected via staining for tartrate resistant acid phosphatase (TRAP; giant, multinucleated, red cells = osteoclasts; indicated by arrows). Asterisks denote significance level of ∗ P
    Figure Legend Snippet: Lack of Thy-1 promotes obesity mediated inflammation. Wildtype (WT) and Thy-1 −/− (KO) mice were fed with a high fat diet for 18 weeks (HFD). (A) Number of adipocytes per total area (N.Adipo/Tt.Ar) and (B) adipocyte area (Adipo.Ar) of adipocytes were analyzed by histology technique. (C) Gene expression of the fat marker fatty-acid-binding protein ( Fabp ) in bone was assessed by RT-PCR. (D) Fat volume (FV/TV) in the femoral medullary cavity was analyzed by osmium tetroxide staining. (E) Representative 3D-images of the fat volume of whole femur. (F,G) Gene expression of the pro-inflammatory markers tumor necrosis factor α ( Tnfα ) and interleukin 6 ( Il6 ). (H) Osteoclast precursor cells from WT mice were cultured ex vivo without (w/o) and with TNFα and osteoclastogenesis was detected via staining for tartrate resistant acid phosphatase (TRAP; giant, multinucleated, red cells = osteoclasts; indicated by arrows). Asterisks denote significance level of ∗ P

    Techniques Used: Mouse Assay, Expressing, Marker, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Staining, Cell Culture, Ex Vivo

    Thy-1 −/− in obesity does not alter the Wnt and YAZ/TAZ pathway. Wildtype (WT) and Thy-1 −/− (KO) mice were fed with a high fat diet for a time period of 18 weeks (HFD). Gene expression of the Wnt pathway inhibitors (A) dickkopf-1 ( Dkk-1 ) and (B) sclerostin ( Sost ) and (C,D) their serum concentrations were evaluated by RT-PCR and ELISA technique, respectively. Gene expression of Wnt ligands such as (E) Wnt5a , (F) 11 , (G) 3a , and (H) 10b in bone was analyzed by RT-PCR. MSCs from WT and Thy-1 −/− mice were treated with TNFα and expression of Yap and Taz of the hippo signaling were investigated. Statistical analysis was performed by (A–H) Student’s t -test and by (I,J) 2-way ANOVA.
    Figure Legend Snippet: Thy-1 −/− in obesity does not alter the Wnt and YAZ/TAZ pathway. Wildtype (WT) and Thy-1 −/− (KO) mice were fed with a high fat diet for a time period of 18 weeks (HFD). Gene expression of the Wnt pathway inhibitors (A) dickkopf-1 ( Dkk-1 ) and (B) sclerostin ( Sost ) and (C,D) their serum concentrations were evaluated by RT-PCR and ELISA technique, respectively. Gene expression of Wnt ligands such as (E) Wnt5a , (F) 11 , (G) 3a , and (H) 10b in bone was analyzed by RT-PCR. MSCs from WT and Thy-1 −/− mice were treated with TNFα and expression of Yap and Taz of the hippo signaling were investigated. Statistical analysis was performed by (A–H) Student’s t -test and by (I,J) 2-way ANOVA.

    Techniques Used: Mouse Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Lack of Thy-1 in obese mice alters the gene expression of RANKL, OPG, and CSF1 under inflammatory conditions. Wildtype (WT) and Thy-1 −/− (KO) mice were fed with a high fat diet for a time period of 18 weeks (HFD). (A) Gene expression of receptor activator of NF-κB ligand (RANKL, Tnfsf11 ), (B) its decoy receptor osteoprotegerin (OPG, Tnfrsf11b ), and (C) receptor of Csf1 (Cfs1r) was analyzed in bone. MSCs from WT and KO mice were treated with TNFα for 24 h to mimic an inflammatory environment and gene expression of Tnfsf11 , Tnfrsf11b , and Cfs1 was determined (D–F) . (G) Summary figure of the key findings. In mice, Thy-1 deficiency results in a reduced osteoclastogenesis and increased adipogenesis leading to a decreased bone formation. Adipocytes produce more of the pro-inflammatory cytokine TNFα and the RANKL-OPG ratio is reduced resulting in an elevated osteoclastogenesis and poor bone mass. Each point represents one mouse and median ± SD is presented. Asterisks denote significance level of ∗ P
    Figure Legend Snippet: Lack of Thy-1 in obese mice alters the gene expression of RANKL, OPG, and CSF1 under inflammatory conditions. Wildtype (WT) and Thy-1 −/− (KO) mice were fed with a high fat diet for a time period of 18 weeks (HFD). (A) Gene expression of receptor activator of NF-κB ligand (RANKL, Tnfsf11 ), (B) its decoy receptor osteoprotegerin (OPG, Tnfrsf11b ), and (C) receptor of Csf1 (Cfs1r) was analyzed in bone. MSCs from WT and KO mice were treated with TNFα for 24 h to mimic an inflammatory environment and gene expression of Tnfsf11 , Tnfrsf11b , and Cfs1 was determined (D–F) . (G) Summary figure of the key findings. In mice, Thy-1 deficiency results in a reduced osteoclastogenesis and increased adipogenesis leading to a decreased bone formation. Adipocytes produce more of the pro-inflammatory cytokine TNFα and the RANKL-OPG ratio is reduced resulting in an elevated osteoclastogenesis and poor bone mass. Each point represents one mouse and median ± SD is presented. Asterisks denote significance level of ∗ P

    Techniques Used: Mouse Assay, Expressing

    19) Product Images from "Comparison of the enzymatic efficiency of Liberase TM and tumor dissociation enzyme: effect on the viability of cells digested from fresh and cryopreserved human ovarian cortex"

    Article Title: Comparison of the enzymatic efficiency of Liberase TM and tumor dissociation enzyme: effect on the viability of cells digested from fresh and cryopreserved human ovarian cortex

    Journal: Reproductive Biology and Endocrinology : RB & E

    doi: 10.1186/s12958-018-0374-6

    Influence of the type of enzymatic treatment of ovarian cortex on the vitality of isolated follicles of different maturity tested applying of Neutral Red dye. a Vitality of follicles in different treatment groups independent of their maturity stage; b Comparison of follicle vitality in Group 1 (fresh ovarian tissues digested with TDE) compared to Group 2 (fresh ovarian tissues digested with Liberase TM) depending on their maturity stage, c Comparison of follicle vitality in Group 3 (frozen ovarian tissues digested with TDE) compared to Group 4 (frozen ovarian tissues digested with Liberase TM) depending on their maturity stage. Bars (mean ± SD) with different superscripts in respective treatment group represent significant differences ( P
    Figure Legend Snippet: Influence of the type of enzymatic treatment of ovarian cortex on the vitality of isolated follicles of different maturity tested applying of Neutral Red dye. a Vitality of follicles in different treatment groups independent of their maturity stage; b Comparison of follicle vitality in Group 1 (fresh ovarian tissues digested with TDE) compared to Group 2 (fresh ovarian tissues digested with Liberase TM) depending on their maturity stage, c Comparison of follicle vitality in Group 3 (frozen ovarian tissues digested with TDE) compared to Group 4 (frozen ovarian tissues digested with Liberase TM) depending on their maturity stage. Bars (mean ± SD) with different superscripts in respective treatment group represent significant differences ( P

    Techniques Used: Isolation

    Influence of the type of enzymatic treatment of ovarian cortex on the vitality of isolated follicles of different maturity tested using of Calcein AM for visualization of viable cells and ethidium homodimer-1 for visualization of dead cells. a Viability of follicles in different treatment groups. b Comparison of follicle vitality in Group 1 (fresh ovarian tissues digested with TDE) compared to Group 2 (fresh ovarian tissues digested with Liberase TM) depending on their maturity stage, c Comparison of follicle vitality in Group 3 (frozen ovarian tissues digested with TDE) compared to Group 4 (frozen ovarian tissues digested with Liberase TM) depending on their maturity stage. Bars (mean ± SD) with different superscripts in respective treatment group represent significant differences ( P
    Figure Legend Snippet: Influence of the type of enzymatic treatment of ovarian cortex on the vitality of isolated follicles of different maturity tested using of Calcein AM for visualization of viable cells and ethidium homodimer-1 for visualization of dead cells. a Viability of follicles in different treatment groups. b Comparison of follicle vitality in Group 1 (fresh ovarian tissues digested with TDE) compared to Group 2 (fresh ovarian tissues digested with Liberase TM) depending on their maturity stage, c Comparison of follicle vitality in Group 3 (frozen ovarian tissues digested with TDE) compared to Group 4 (frozen ovarian tissues digested with Liberase TM) depending on their maturity stage. Bars (mean ± SD) with different superscripts in respective treatment group represent significant differences ( P

    Techniques Used: Isolation

    Typical view of follicle suspension after enzymatic digestion. a follicles isolated from frozen ovarian cortex with TDE-enzyme cocktail; b follicles isolated from frozen ovarian cortex with Liberase TM. The black arrows show the clustered and partially isolated from incompletely digested stroma follicles. Bar = 50 μm
    Figure Legend Snippet: Typical view of follicle suspension after enzymatic digestion. a follicles isolated from frozen ovarian cortex with TDE-enzyme cocktail; b follicles isolated from frozen ovarian cortex with Liberase TM. The black arrows show the clustered and partially isolated from incompletely digested stroma follicles. Bar = 50 μm

    Techniques Used: Isolation

    20) Product Images from "Ebi3 Prevents Trypanosoma cruzi-Induced Myocarditis by Dampening IFN-γ-Driven Inflammation"

    Article Title: Ebi3 Prevents Trypanosoma cruzi-Induced Myocarditis by Dampening IFN-γ-Driven Inflammation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01213

    IL-27 is highly produced by myeloid cells following infection with Trypanosoma cruzi . (A,B) Dendritic cells and macrophages were differentiated from bone marrows of naïve wild-type (WT) mice and stimulated with medium or T. cruzi trypomastigotes (MOI 3:1). After 48 h, supernatant was collected and IL-27p28 levels were assessed by ELISA. (C,D) Splenocytes from naïve WT were cultured with medium or trypomastigotes (MOI = 3:1) for 6 or 48 h for mRNA or protein quantification of IL-27. (E,F) Intracellular expression of IL-27p28 by MHCII + CD11b + myeloid cells 15 days after T. cruzi infection. Hearts of the WT mice infected or not were collected and digested with a cocktail of liberase TL (Roche). Cells were re-stimulated in vitro with PMA/ionomycin for 6 h and subjected to extracellular staining for MHCII and CD11b and intracellular staining for IL-27p28. Data are expressed as representative dot plots, percentage, and absolute number of IL-27 + MHCII + CD11b + cells. Data shown are mean ± SEM of cultured cells in triplicates and from five mice per group and are representative of two independent experiments.
    Figure Legend Snippet: IL-27 is highly produced by myeloid cells following infection with Trypanosoma cruzi . (A,B) Dendritic cells and macrophages were differentiated from bone marrows of naïve wild-type (WT) mice and stimulated with medium or T. cruzi trypomastigotes (MOI 3:1). After 48 h, supernatant was collected and IL-27p28 levels were assessed by ELISA. (C,D) Splenocytes from naïve WT were cultured with medium or trypomastigotes (MOI = 3:1) for 6 or 48 h for mRNA or protein quantification of IL-27. (E,F) Intracellular expression of IL-27p28 by MHCII + CD11b + myeloid cells 15 days after T. cruzi infection. Hearts of the WT mice infected or not were collected and digested with a cocktail of liberase TL (Roche). Cells were re-stimulated in vitro with PMA/ionomycin for 6 h and subjected to extracellular staining for MHCII and CD11b and intracellular staining for IL-27p28. Data are expressed as representative dot plots, percentage, and absolute number of IL-27 + MHCII + CD11b + cells. Data shown are mean ± SEM of cultured cells in triplicates and from five mice per group and are representative of two independent experiments.

    Techniques Used: Produced, Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Expressing, In Vitro, Staining

    21) Product Images from "Ageing compromises mouse thymus function and remodels epithelial cell differentiation"

    Article Title: Ageing compromises mouse thymus function and remodels epithelial cell differentiation

    Journal: bioRxiv

    doi: 10.1101/2020.03.02.973008

    Quality control and summary of multiplexe d single-cell droplet RNA sequencing. (a) FAC-Sorting gating strategy for the isolation of ZsGreen +/− TEC subpopula tions. (b) Multiplet detection using multiplexe d hashtag oligos (HTO). Coloured bars denotes the number of cells in each sample (Chromium chip well), where either no (Dropout), multiple (Multiplet) or a single HTO was detected in a droplet. (c) The distribution of singlet cells across samples and experimental conditions (age and ZsGreen fraction). (d) A mapping of single-cell clusters onto equivalent ageing clusters. (e) Uniform manifold approximation and projection (UMAP). Points are single cells coloured by the assigned TEC subtype. Cells are split into panels based on the age of the mouse at the time of doxycycline treatment. (f) A UMAP split by mouse age showing the estimated deconvolution size factors. The boxplot on the right shows the distribution of size factors across single-cell clusters. (g) The number of detected genes (log expression > 0) in single cells overlaid on a UMAP and split by age. The boxplots on the right-hand side show the distributions of the number of detected genes for each single cell cluster.
    Figure Legend Snippet: Quality control and summary of multiplexe d single-cell droplet RNA sequencing. (a) FAC-Sorting gating strategy for the isolation of ZsGreen +/− TEC subpopula tions. (b) Multiplet detection using multiplexe d hashtag oligos (HTO). Coloured bars denotes the number of cells in each sample (Chromium chip well), where either no (Dropout), multiple (Multiplet) or a single HTO was detected in a droplet. (c) The distribution of singlet cells across samples and experimental conditions (age and ZsGreen fraction). (d) A mapping of single-cell clusters onto equivalent ageing clusters. (e) Uniform manifold approximation and projection (UMAP). Points are single cells coloured by the assigned TEC subtype. Cells are split into panels based on the age of the mouse at the time of doxycycline treatment. (f) A UMAP split by mouse age showing the estimated deconvolution size factors. The boxplot on the right shows the distribution of size factors across single-cell clusters. (g) The number of detected genes (log expression > 0) in single cells overlaid on a UMAP and split by age. The boxplots on the right-hand side show the distributions of the number of detected genes for each single cell cluster.

    Techniques Used: RNA Sequencing Assay, Isolation, Chromatin Immunoprecipitation, Expressing

    Ageing restricts the differentiation of intertypical TEC into mature mTEC. (a) RNA velocity estimates overlaid on a uniform manifold approximation and projection (UMAP) of all single cells across all ages derived from 3xtg β5t mice. Cells are coloured by annotated clusters ( Supplementary Figure 8 ) defined using a random-walk on an SNN-graph (Methods). Annotations were assigned based on the co-expression of key marker genes ( Supplementary Figures 9 10 ). Inset panel: schematic representation of ZsG lineage tracing of TEC across mouse ages. Paired colour arrows denote the time and age of doxycycline treatment. Numbers above the arrows represent the age of mice at the time of single-cell measurements. (b) Differential abundance testing of TEC clusters from (a) across age and between lineage tracing fractions. The volcano plot shows the log fold change (LFC; x-axis) against −log 10 FDR (y-axis) of the interaction between lineage fraction and age. TEC clusters that have significantly different changes in the ZsG+ compared to ZsG-fraction over age (FDR 5%) are coloured in red and labelled. Positive log-fold changes represent a higher rate of change over age in the ZsG+ fraction, whilst negative log-fold changes represent a higher rate of change in the ZsG-fraction. (c) Individual best-fit line plots show the sub-cluster frequency (y-axis) at each dox-treatment age (x-axis), grouped and coloured by ZsG fraction. The shaded band represents the linear model 95% confidence interval around the linear fit. (d) Boxplot of Psmb11 single-cell expression (log 10 normalised counts) across 4 intertypical TEC clusters, coloured by age at time of dox-treatment.
    Figure Legend Snippet: Ageing restricts the differentiation of intertypical TEC into mature mTEC. (a) RNA velocity estimates overlaid on a uniform manifold approximation and projection (UMAP) of all single cells across all ages derived from 3xtg β5t mice. Cells are coloured by annotated clusters ( Supplementary Figure 8 ) defined using a random-walk on an SNN-graph (Methods). Annotations were assigned based on the co-expression of key marker genes ( Supplementary Figures 9 10 ). Inset panel: schematic representation of ZsG lineage tracing of TEC across mouse ages. Paired colour arrows denote the time and age of doxycycline treatment. Numbers above the arrows represent the age of mice at the time of single-cell measurements. (b) Differential abundance testing of TEC clusters from (a) across age and between lineage tracing fractions. The volcano plot shows the log fold change (LFC; x-axis) against −log 10 FDR (y-axis) of the interaction between lineage fraction and age. TEC clusters that have significantly different changes in the ZsG+ compared to ZsG-fraction over age (FDR 5%) are coloured in red and labelled. Positive log-fold changes represent a higher rate of change over age in the ZsG+ fraction, whilst negative log-fold changes represent a higher rate of change in the ZsG-fraction. (c) Individual best-fit line plots show the sub-cluster frequency (y-axis) at each dox-treatment age (x-axis), grouped and coloured by ZsG fraction. The shaded band represents the linear model 95% confidence interval around the linear fit. (d) Boxplot of Psmb11 single-cell expression (log 10 normalised counts) across 4 intertypical TEC clusters, coloured by age at time of dox-treatment.

    Techniques Used: Derivative Assay, Mouse Assay, Expressing, Marker

    Thymic stromal remodelling during ageing. (a) A schematic showing the experimental design and FACS phenotypes of sorted cells for single-cell RNA-sequencing. Right panel shows cell composition fluctuations as a relative fraction of all EpCAM+ TEC with respect to the TEC subsets investigated. Remaining EpCAM+ cells not FAC-sorted are represented in the EpCAM+ population. (b) A SPRING-layout of the shared nearest-neighbour graph of single TEC, derived from scRNA-seq transcriptional profiles. Graph nodes represent single cells and edges represent shared k-nearest neighbours (k=5). Cells are coloured by a clustering that joins highly connected networks of cells based on a random walk (Walktrap ( Pons and Latapy, 2005 )). Clusters are annotated based on comparisons to known TEC subsets and stereotypical expression profiles ( Table 1 ). (c) A heatmap of marker genes for TEC subtypes identified from single-cell transcriptome profiling annotated as in (b). (d) Enrichment of MSigDB biological pathways with age in mature cTEC, intertypical TEC and mature mTEC, annotated as in (b). Bars denote normalised enrichment score (NES) for significant pathways (FDR 5%), with enrichments coloured by cell type. Age-related alterations are shown in the context of pathways that are up-regulated (left), down-regulated (right) or do not change (middle) across multiple tissues and species ( Benayoun et al., 2019 ). (e) A ribbon-plot demonstrating the compositional changes in TEC subtypes across ages, as an estimated fraction of all TEC (EpCAM+). Colours indicating each subtype are shown above the plot with unsorted TEC indicated in white. (f) A volcano-plot of a negative binomial generalised linear model (GLM) showing linear (left) and quadratic (right) changes in cell cluster abundance as a function of age. X-axis denotes the change (Δ) in cellularity per week, and the Y-axis shows the −log 10 false discovery rate (FDR). Subtypes with statistical evidence of abundance changes (FDR 1%) are labelled and shown as red points.
    Figure Legend Snippet: Thymic stromal remodelling during ageing. (a) A schematic showing the experimental design and FACS phenotypes of sorted cells for single-cell RNA-sequencing. Right panel shows cell composition fluctuations as a relative fraction of all EpCAM+ TEC with respect to the TEC subsets investigated. Remaining EpCAM+ cells not FAC-sorted are represented in the EpCAM+ population. (b) A SPRING-layout of the shared nearest-neighbour graph of single TEC, derived from scRNA-seq transcriptional profiles. Graph nodes represent single cells and edges represent shared k-nearest neighbours (k=5). Cells are coloured by a clustering that joins highly connected networks of cells based on a random walk (Walktrap ( Pons and Latapy, 2005 )). Clusters are annotated based on comparisons to known TEC subsets and stereotypical expression profiles ( Table 1 ). (c) A heatmap of marker genes for TEC subtypes identified from single-cell transcriptome profiling annotated as in (b). (d) Enrichment of MSigDB biological pathways with age in mature cTEC, intertypical TEC and mature mTEC, annotated as in (b). Bars denote normalised enrichment score (NES) for significant pathways (FDR 5%), with enrichments coloured by cell type. Age-related alterations are shown in the context of pathways that are up-regulated (left), down-regulated (right) or do not change (middle) across multiple tissues and species ( Benayoun et al., 2019 ). (e) A ribbon-plot demonstrating the compositional changes in TEC subtypes across ages, as an estimated fraction of all TEC (EpCAM+). Colours indicating each subtype are shown above the plot with unsorted TEC indicated in white. (f) A volcano-plot of a negative binomial generalised linear model (GLM) showing linear (left) and quadratic (right) changes in cell cluster abundance as a function of age. X-axis denotes the change (Δ) in cellularity per week, and the Y-axis shows the −log 10 false discovery rate (FDR). Subtypes with statistical evidence of abundance changes (FDR 1%) are labelled and shown as red points.

    Techniques Used: FACS, RNA Sequencing Assay, Derivative Assay, Expressing, Marker

    Experimental investigation of the ageing thymus. (a) FACS gating strategy for isolation of TEC sort types. (b) Filtering strategy to identify high-quality TEC libraries. (c-f) Fractions of libraries filtered out based on sparsity threshold (c), the fraction of reads from mitochondrial genes expressed (d), library size thresholds (e), or ERCC-spike in RNA % expression threshold (f). (g) Sparsity in each single cell cluster by age. (h) Reassignment of libraries to clusters based on downsampling fraction. Excessive downsampling leads to an accumulation of the low-diversity library.
    Figure Legend Snippet: Experimental investigation of the ageing thymus. (a) FACS gating strategy for isolation of TEC sort types. (b) Filtering strategy to identify high-quality TEC libraries. (c-f) Fractions of libraries filtered out based on sparsity threshold (c), the fraction of reads from mitochondrial genes expressed (d), library size thresholds (e), or ERCC-spike in RNA % expression threshold (f). (g) Sparsity in each single cell cluster by age. (h) Reassignment of libraries to clusters based on downsampling fraction. Excessive downsampling leads to an accumulation of the low-diversity library.

    Techniques Used: FACS, Isolation, RNA Expression

    22) Product Images from "Optimization of Liposomes for Antigen Targeting to Splenic CD169+ Macrophages"

    Article Title: Optimization of Liposomes for Antigen Targeting to Splenic CD169+ Macrophages

    Journal: Pharmaceutics

    doi: 10.3390/pharmaceutics12121138

    Liposomal incorporation of PEG abolishes the interaction between GM3 and CD169. ( A ) Liposomes were coated overnight on an ELISA plate, and binding to WT or mutant CD169 Fc was quantified. Indicated is the average OD450 of 3 independent experiments. ( B – D ) DiD-containing liposomes were incubated with TSn at 4 or 37 °C for 45 min. Representative dot plots following incubation at 37 °C (20 µM liposomes) ( B ). Indicated is the average GMFI ± SD of technical triplicates after incubation at 4 °C (C) and at 37 °C ( D ) (pattern representative of 4 independent experiments). ( E ) DiD-containing liposomes were incubated for 45 min at 37 °C with freshly isolated splenocytes from C57BL/6 WT or W2QR97A-mutant CD169 mice. Subsequently, DiD fluorescence was quantified for distinct cell populations using flow cytometry. Indicated is the GMFI ± SD ( n = 5) (representative of 2 independent experiments). ( F ) DiD-containing liposomes (90 nmol of phospholipid), supplemented with adjuvant, were injected IV in mice, and after 2 h, splenic cell populations were analyzed for DiD fluorescence. Indicated is the average GMFI ± SD ( n = 4). *** p
    Figure Legend Snippet: Liposomal incorporation of PEG abolishes the interaction between GM3 and CD169. ( A ) Liposomes were coated overnight on an ELISA plate, and binding to WT or mutant CD169 Fc was quantified. Indicated is the average OD450 of 3 independent experiments. ( B – D ) DiD-containing liposomes were incubated with TSn at 4 or 37 °C for 45 min. Representative dot plots following incubation at 37 °C (20 µM liposomes) ( B ). Indicated is the average GMFI ± SD of technical triplicates after incubation at 4 °C (C) and at 37 °C ( D ) (pattern representative of 4 independent experiments). ( E ) DiD-containing liposomes were incubated for 45 min at 37 °C with freshly isolated splenocytes from C57BL/6 WT or W2QR97A-mutant CD169 mice. Subsequently, DiD fluorescence was quantified for distinct cell populations using flow cytometry. Indicated is the GMFI ± SD ( n = 5) (representative of 2 independent experiments). ( F ) DiD-containing liposomes (90 nmol of phospholipid), supplemented with adjuvant, were injected IV in mice, and after 2 h, splenic cell populations were analyzed for DiD fluorescence. Indicated is the average GMFI ± SD ( n = 4). *** p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay, Mutagenesis, Incubation, Isolation, Mouse Assay, Fluorescence, Flow Cytometry, Injection

    Inclusion of GM3 in liposomes results in specific binding to CD169-expressing cells in vitro. ( A ) Liposomes were coated overnight on an ELISA plate, and binding to wild-type (WT) or mutant mouse CD169 Fc was quantified. Indicated is the average OD450 of 3 independent experiments. ( B – D ) 1′-dioctadecyl-3,3,3′,3′-tetramethyl indodicarbocyanine (DiD)-containing liposomes were incubated with TSn at 4 or 37 °C for 45 min. Representative dot plots following incubation on 37 °C (20 µM liposomes) ( B ). Indicated is the average geometric mean fluorescence intensity GMFI ± SD of a technical triplicate after incubation at 4 °C ( C ) and at 37 °C ( D ) (pattern representative of 4 independent experiments). ( E ) DiD-containing liposomes were incubated for 45 min at 37 °C with freshly isolated splenocytes from C57BL/6 WT or W2QR97A-mutant CD169 mice. Subsequently, DiD fluorescence was quantified for distinct cell populations using flow cytometry. Indicated is the GMFI ± SD ( n = 5) (representative of 2 independent experiments). * p
    Figure Legend Snippet: Inclusion of GM3 in liposomes results in specific binding to CD169-expressing cells in vitro. ( A ) Liposomes were coated overnight on an ELISA plate, and binding to wild-type (WT) or mutant mouse CD169 Fc was quantified. Indicated is the average OD450 of 3 independent experiments. ( B – D ) 1′-dioctadecyl-3,3,3′,3′-tetramethyl indodicarbocyanine (DiD)-containing liposomes were incubated with TSn at 4 or 37 °C for 45 min. Representative dot plots following incubation on 37 °C (20 µM liposomes) ( B ). Indicated is the average geometric mean fluorescence intensity GMFI ± SD of a technical triplicate after incubation at 4 °C ( C ) and at 37 °C ( D ) (pattern representative of 4 independent experiments). ( E ) DiD-containing liposomes were incubated for 45 min at 37 °C with freshly isolated splenocytes from C57BL/6 WT or W2QR97A-mutant CD169 mice. Subsequently, DiD fluorescence was quantified for distinct cell populations using flow cytometry. Indicated is the GMFI ± SD ( n = 5) (representative of 2 independent experiments). * p

    Techniques Used: Binding Assay, Expressing, In Vitro, Enzyme-linked Immunosorbent Assay, Mutagenesis, Incubation, Fluorescence, Isolation, Mouse Assay, Flow Cytometry

    Size of GM3-containing liposomes influences binding to CD169-expressing cells in vivo, but not in vitro. ( A ) Liposomes were coated overnight on an ELISA plate, and binding to WT or mutant CD169 Fc was quantified. Indicated is the average OD450 of 2 independent technical triplicates. ( B – D ) DiD-containing liposomes were incubated with TSn at 4 or 37 °C for 45 min. Representative dot plots following incubation at 37 °C (20 µM liposomes) ( B ). Indicated is the average GMFI ± SD of a technical triplicate after incubation at 4 °C ( C ) and at 37 °C ( D ) (representative of 4 independent experiments). ( E ) DiD-containing liposomes were incubated for 45 min at 37 °C with freshly isolated splenocytes from C57BL/6 WT mice. Subsequently, DiD fluorescence was quantified for distinct cell populations using flow cytometry. Indicated is the GMFI ± SD ( n = 4) (representative of 2 independent experiments). ( F ) DiD-containing liposomes (22.5 nmol of phospholipid) were injected IV in mice, and after 2 h, splenic cell populations were analyzed for DiD fluorescence. Indicated is the average GMFI ± SD ( n = 4) ( F ). * p
    Figure Legend Snippet: Size of GM3-containing liposomes influences binding to CD169-expressing cells in vivo, but not in vitro. ( A ) Liposomes were coated overnight on an ELISA plate, and binding to WT or mutant CD169 Fc was quantified. Indicated is the average OD450 of 2 independent technical triplicates. ( B – D ) DiD-containing liposomes were incubated with TSn at 4 or 37 °C for 45 min. Representative dot plots following incubation at 37 °C (20 µM liposomes) ( B ). Indicated is the average GMFI ± SD of a technical triplicate after incubation at 4 °C ( C ) and at 37 °C ( D ) (representative of 4 independent experiments). ( E ) DiD-containing liposomes were incubated for 45 min at 37 °C with freshly isolated splenocytes from C57BL/6 WT mice. Subsequently, DiD fluorescence was quantified for distinct cell populations using flow cytometry. Indicated is the GMFI ± SD ( n = 4) (representative of 2 independent experiments). ( F ) DiD-containing liposomes (22.5 nmol of phospholipid) were injected IV in mice, and after 2 h, splenic cell populations were analyzed for DiD fluorescence. Indicated is the average GMFI ± SD ( n = 4) ( F ). * p

    Techniques Used: Binding Assay, Expressing, In Vivo, In Vitro, Enzyme-linked Immunosorbent Assay, Mutagenesis, Incubation, Isolation, Mouse Assay, Fluorescence, Flow Cytometry, Injection

    23) Product Images from "Structure of a patient-derived antibody in complex with allergen reveals simultaneous conventional and superantigen-like recognition"

    Article Title: Structure of a patient-derived antibody in complex with allergen reveals simultaneous conventional and superantigen-like recognition

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1806840115

    Characterization of oligomeric state and basophil degranulation for the three polcalcin allergens. ( A ) Chromatograms of the RI signals for Phl p 7, Bet v 4, and Ole e 3. The three proteins [in 25 mM Tris⋅HCl (pH 7.4) and 150 mM NaCl with 4 mM CaCl 2 ] were run over a Superdex 75 5/150 column (GE Healthcare) at a flow rate of 0.3 mL/min. The differential RI is shown in black (A.U., arbitrary units). The green lines across the protein elution volumes show the molecular masses of the proteins, indicating a monomeric state. ( B ) Basophil degranulation assay showing the percentage degranulation of RBL-SX38 cells sensitized with 102.1F10 IgE (black bars) and CS09G6K IgE (white bars) antibodies followed by stimulation with 5,000 ng/μL of Phl p 7, Bet v 4, or Ole e 3 or with assay medium (background). Degranulation is expressed as a percentage of that induced by cell lysis. Bars represent the mean ± SEM of three independent experiments. Statistical significance was determined by t test; **** P
    Figure Legend Snippet: Characterization of oligomeric state and basophil degranulation for the three polcalcin allergens. ( A ) Chromatograms of the RI signals for Phl p 7, Bet v 4, and Ole e 3. The three proteins [in 25 mM Tris⋅HCl (pH 7.4) and 150 mM NaCl with 4 mM CaCl 2 ] were run over a Superdex 75 5/150 column (GE Healthcare) at a flow rate of 0.3 mL/min. The differential RI is shown in black (A.U., arbitrary units). The green lines across the protein elution volumes show the molecular masses of the proteins, indicating a monomeric state. ( B ) Basophil degranulation assay showing the percentage degranulation of RBL-SX38 cells sensitized with 102.1F10 IgE (black bars) and CS09G6K IgE (white bars) antibodies followed by stimulation with 5,000 ng/μL of Phl p 7, Bet v 4, or Ole e 3 or with assay medium (background). Degranulation is expressed as a percentage of that induced by cell lysis. Bars represent the mean ± SEM of three independent experiments. Statistical significance was determined by t test; **** P

    Techniques Used: Flow Cytometry, Degranulation Assay, Lysis

    24) Product Images from "Combined CSL and p53 downregulation promotes cancer-associated fibroblast activation"

    Article Title: Combined CSL and p53 downregulation promotes cancer-associated fibroblast activation

    Journal: Nature cell biology

    doi: 10.1038/ncb3228

    CSL expression and function in CAFs (a) Immunoblotting of cancer associated fibroblasts (CAFs) from several skin SCCs soon after culturing (p2) and upon expansion (p4; *) in parallel with several HDF strains. Blot was probed for CSL, α-SMAand γ-tubulin with densitometric quantification; n(strains)= 5 HDF, 4 CAF, 5 CAF*, mean +/− s.e.m., two-tailed unpaired t-test. Similar results were obtained in a second independent experiment (right panel). (b) Same CAFs strains as in (a), transduced with lentiviral vector fordoxycyclin induciblemyc-tagged CSL or pIND vector control were analyzed by immunoblotting. (c) CAFs as in (b) were analyzed by RT-qPCR for indicated genes; n(CAF strains)=3 pInd-Ctrl, 3 pInd-CSL, ratio(CSL/Ctrl), two-tailed one sample t-tets. (d) DsRed2-expressing SCC13 cells admixed with CAFs (strain #2) plus/minus constitutive lentiviral CSL expression were injected in parallel into ears of 3 NOD/SCID Il2rg−/− 10-weeks-old male mice. Representative images and signal quantification relative to day 1; n=3 per condition, mean +/− s.e.m., two-tailed paired t-test at day 10. (e) Immunofluorescence for proliferation (phospho-histone 3) and epithelial (pankeratin) markers. (f) CSL levels in published gene expression profiles of CAFs from skin SCCs (from general populations (cSCC) and from patients with recessive dystrophic epidermolysis bullosa (RDEB-SCC); head/neck SCC, breast cancer and lung cancer; Skin (n=5 SCC, 4 RDEB-SCC, 3 healthy individuals), Head Neck (n=7 SCC, 5 healthy individuals), Breast (n=23 carcinoma, 5 healthy individuals), Lung (n=4 NSCLC, 15 healthy individuals), two-class comparison with moderated t-statistic. Median, upper and lower quartiles are represented. Vertical whiskers indicate variability outside the upper and lower quartiles. (g) Heat maps of differentially expressed genes in HDFs plus/minus CSL silencing relative to data sets of CAFs from skin and head/neck SCCs. Genes modulated by CSL silencing in HDFs ( > 1.4 folds) concordantly or discordantly down- or up-regulated in clinically occurring CAFs are indicated by dark and light turquoise and magenta colors, respectively. Selected pathways or processes with a statistically significant enrichment (p
    Figure Legend Snippet: CSL expression and function in CAFs (a) Immunoblotting of cancer associated fibroblasts (CAFs) from several skin SCCs soon after culturing (p2) and upon expansion (p4; *) in parallel with several HDF strains. Blot was probed for CSL, α-SMAand γ-tubulin with densitometric quantification; n(strains)= 5 HDF, 4 CAF, 5 CAF*, mean +/− s.e.m., two-tailed unpaired t-test. Similar results were obtained in a second independent experiment (right panel). (b) Same CAFs strains as in (a), transduced with lentiviral vector fordoxycyclin induciblemyc-tagged CSL or pIND vector control were analyzed by immunoblotting. (c) CAFs as in (b) were analyzed by RT-qPCR for indicated genes; n(CAF strains)=3 pInd-Ctrl, 3 pInd-CSL, ratio(CSL/Ctrl), two-tailed one sample t-tets. (d) DsRed2-expressing SCC13 cells admixed with CAFs (strain #2) plus/minus constitutive lentiviral CSL expression were injected in parallel into ears of 3 NOD/SCID Il2rg−/− 10-weeks-old male mice. Representative images and signal quantification relative to day 1; n=3 per condition, mean +/− s.e.m., two-tailed paired t-test at day 10. (e) Immunofluorescence for proliferation (phospho-histone 3) and epithelial (pankeratin) markers. (f) CSL levels in published gene expression profiles of CAFs from skin SCCs (from general populations (cSCC) and from patients with recessive dystrophic epidermolysis bullosa (RDEB-SCC); head/neck SCC, breast cancer and lung cancer; Skin (n=5 SCC, 4 RDEB-SCC, 3 healthy individuals), Head Neck (n=7 SCC, 5 healthy individuals), Breast (n=23 carcinoma, 5 healthy individuals), Lung (n=4 NSCLC, 15 healthy individuals), two-class comparison with moderated t-statistic. Median, upper and lower quartiles are represented. Vertical whiskers indicate variability outside the upper and lower quartiles. (g) Heat maps of differentially expressed genes in HDFs plus/minus CSL silencing relative to data sets of CAFs from skin and head/neck SCCs. Genes modulated by CSL silencing in HDFs ( > 1.4 folds) concordantly or discordantly down- or up-regulated in clinically occurring CAFs are indicated by dark and light turquoise and magenta colors, respectively. Selected pathways or processes with a statistically significant enrichment (p

    Techniques Used: Expressing, Two Tailed Test, Transduction, Plasmid Preparation, Quantitative RT-PCR, Injection, Mouse Assay, Immunofluorescence

    25) Product Images from "Breg dependent islet transplant tolerance is also NK-cell dependent"

    Article Title: Breg dependent islet transplant tolerance is also NK-cell dependent

    Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

    doi: 10.1111/ajt.14265

    Antibody induced islet transplant tolerance is associated with skewing of NK1.1 + cells B6 recipients of Balb/c Islet grafts were rendered tolerant by dual antibody treatment (n=4). 16 days post-transplant, whole splenocytes were isolated for immunophenotyping by flow cytometry. The amount of NK cells was reduced (2.07% vs. 3.8% NK1.1 + , DX5 + , CD3 − ; p=0.0022) and proportion of NK-T cells increased by nearly 50 % as compared to naïve control animals (2.46% vs. 1.3% NK1.1 + , CD3 + ; p=0.0093).
    Figure Legend Snippet: Antibody induced islet transplant tolerance is associated with skewing of NK1.1 + cells B6 recipients of Balb/c Islet grafts were rendered tolerant by dual antibody treatment (n=4). 16 days post-transplant, whole splenocytes were isolated for immunophenotyping by flow cytometry. The amount of NK cells was reduced (2.07% vs. 3.8% NK1.1 + , DX5 + , CD3 − ; p=0.0022) and proportion of NK-T cells increased by nearly 50 % as compared to naïve control animals (2.46% vs. 1.3% NK1.1 + , CD3 + ; p=0.0093).

    Techniques Used: Isolation, Flow Cytometry, Cytometry

    Antibody depletes host NK-cells without altering Treg and Breg populations (A+B) NK1.1 Antibody treatment thoroughly depletes NK-cells in whole splenocytes (n=3 B6 mice). Splenocytes were harvested 16 days post Balb/c islet transplant and 8 days after last dose of anti-NK1.1 in mice receiving dual Ab treatment. (p
    Figure Legend Snippet: Antibody depletes host NK-cells without altering Treg and Breg populations (A+B) NK1.1 Antibody treatment thoroughly depletes NK-cells in whole splenocytes (n=3 B6 mice). Splenocytes were harvested 16 days post Balb/c islet transplant and 8 days after last dose of anti-NK1.1 in mice receiving dual Ab treatment. (p

    Techniques Used: Mouse Assay

    26) Product Images from "Biologically indeterminate yet ordered promiscuous gene expression in single medullary thymic epithelial cells"

    Article Title: Biologically indeterminate yet ordered promiscuous gene expression in single medullary thymic epithelial cells

    Journal: The EMBO Journal

    doi: 10.15252/embj.2019101828

    Deep transcriptome analysis at single‐cell resolution of thousands of flow cytometrically sorted mTEC mTEC promiscuously expressing TSPAN8 and/or GP2 (upper right panel/red) on their cell surface can be identified by flow cytometry (only final gates are shown: see Appendix Fig S1 for full gating strategy). mTEC were identified as CD45 − EpCAM + Ly51 − ( Appendix Fig S1 ) and the gates for TSPAN8/GP2 were set against isotype control antibodies (left panels/grey). Lower right panel: bar graph showing mean frequency (±SD) of TSPAN8 + , GP2 + , and TSPAN8 + GP2 + cells within total mTEC; results represent pooled data from 3 (TSPAN8 + ), 4 (GP2 + ) and 2 (TSPAN8 + GP2 + ) independent experiments each containing three individual mice. Identification of TSPAN8 or GP2 protein expression via FACS reflects mRNA expression. Bar graph showing mean expression (±SD) of Tspan8 and Gp2 mRNA relative to β‐actin by RT–qPCR on FACS sorted mTEC negative or positive for TSPAN8 or GP2 protein, respectively; n = 3, representative of two independent experiments. Significance by Students t ‐test; *** P
    Figure Legend Snippet: Deep transcriptome analysis at single‐cell resolution of thousands of flow cytometrically sorted mTEC mTEC promiscuously expressing TSPAN8 and/or GP2 (upper right panel/red) on their cell surface can be identified by flow cytometry (only final gates are shown: see Appendix Fig S1 for full gating strategy). mTEC were identified as CD45 − EpCAM + Ly51 − ( Appendix Fig S1 ) and the gates for TSPAN8/GP2 were set against isotype control antibodies (left panels/grey). Lower right panel: bar graph showing mean frequency (±SD) of TSPAN8 + , GP2 + , and TSPAN8 + GP2 + cells within total mTEC; results represent pooled data from 3 (TSPAN8 + ), 4 (GP2 + ) and 2 (TSPAN8 + GP2 + ) independent experiments each containing three individual mice. Identification of TSPAN8 or GP2 protein expression via FACS reflects mRNA expression. Bar graph showing mean expression (±SD) of Tspan8 and Gp2 mRNA relative to β‐actin by RT–qPCR on FACS sorted mTEC negative or positive for TSPAN8 or GP2 protein, respectively; n = 3, representative of two independent experiments. Significance by Students t ‐test; *** P

    Techniques Used: Flow Cytometry, Expressing, Cytometry, Mouse Assay, FACS, Quantitative RT-PCR

    27) Product Images from "Wheat germ agglutinin–conjugated fluorescent pH sensors for visualizing proton fluxes"

    Article Title: Wheat germ agglutinin–conjugated fluorescent pH sensors for visualizing proton fluxes

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.201912498

    Structured light microscopy of lactate-stimulated proton transport in rat ventricular myocytes labeled with WGA-pHRho. (A) A ∼750-nm illuminated z -plane of a WGA-pHRho–labeled ventricular myocyte ∼2 μm above the coverslip focal plane. (B) Time course of 10 mM lactate perfusion before (red) and after addition of 50 nM AR-C155858 (gray). Plotted data are mean + SEM (shading) for clarity ( n = 6 cells); data were collected at 1 Hz. (C) Pseudocolor image depicting the eroding pixel (5 px) analysis to generate the four annuli. (D) Changes of ΔF/F 0 before, during, and after perfusion of 10 mM lactate at each annulus (A0–A3). (E) Pixel intensity histogram of the four annuli (area normalized) for cell shown in A. (F) Average maximal |ΔF/F 0 | upon wash in (solid bars) and wash out (open bars) of 10 mM lactate for nine cells. One-way ANOVA (Bonferroni's multiple comparison test) was used to determine significance (*, P
    Figure Legend Snippet: Structured light microscopy of lactate-stimulated proton transport in rat ventricular myocytes labeled with WGA-pHRho. (A) A ∼750-nm illuminated z -plane of a WGA-pHRho–labeled ventricular myocyte ∼2 μm above the coverslip focal plane. (B) Time course of 10 mM lactate perfusion before (red) and after addition of 50 nM AR-C155858 (gray). Plotted data are mean + SEM (shading) for clarity ( n = 6 cells); data were collected at 1 Hz. (C) Pseudocolor image depicting the eroding pixel (5 px) analysis to generate the four annuli. (D) Changes of ΔF/F 0 before, during, and after perfusion of 10 mM lactate at each annulus (A0–A3). (E) Pixel intensity histogram of the four annuli (area normalized) for cell shown in A. (F) Average maximal |ΔF/F 0 | upon wash in (solid bars) and wash out (open bars) of 10 mM lactate for nine cells. One-way ANOVA (Bonferroni's multiple comparison test) was used to determine significance (*, P

    Techniques Used: Light Microscopy, Labeling, Whole Genome Amplification

    28) Product Images from "Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue"

    Article Title: Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-018-1631-y

    High synovial cell yield, preserved surface markers, and reproducible transcriptomic results with mechanical and enzymatic disaggregation protocol. a Total cell counts per gram from synovial tissue mechanically disaggregated with or without Liberase™ TL proteolytic enzyme treatment. n = 16, paired t test. b , c Flow cytometry detection and quantification of stromal cells (CD45 – PDPN + ) upon mechanical disaggregation with or without Liberase™ TL proteolytic enzyme treatment. d Flow cytometry of dissociated synovial cells treated with panel of proteolytic Liberase™ TL formulations. Cells gated for viability and CD3 T-cell receptor subunit. Plots representative of n = 4 biologic replicates. e Total RNA yield from synovial tissue dissociated with three concentrations of Liberase™ TL. Two or three technical replicates from nine tissues used for each enzyme concentration. ANOVA with Tukey's comparison, * p
    Figure Legend Snippet: High synovial cell yield, preserved surface markers, and reproducible transcriptomic results with mechanical and enzymatic disaggregation protocol. a Total cell counts per gram from synovial tissue mechanically disaggregated with or without Liberase™ TL proteolytic enzyme treatment. n = 16, paired t test. b , c Flow cytometry detection and quantification of stromal cells (CD45 – PDPN + ) upon mechanical disaggregation with or without Liberase™ TL proteolytic enzyme treatment. d Flow cytometry of dissociated synovial cells treated with panel of proteolytic Liberase™ TL formulations. Cells gated for viability and CD3 T-cell receptor subunit. Plots representative of n = 4 biologic replicates. e Total RNA yield from synovial tissue dissociated with three concentrations of Liberase™ TL. Two or three technical replicates from nine tissues used for each enzyme concentration. ANOVA with Tukey's comparison, * p

    Techniques Used: Flow Cytometry, Cytometry, Concentration Assay

    29) Product Images from "Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue"

    Article Title: Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-018-1631-y

    High synovial cell yield, preserved surface markers, and reproducible transcriptomic results with mechanical and enzymatic disaggregation protocol. a Total cell counts per gram from synovial tissue mechanically disaggregated with or without Liberase™ TL proteolytic enzyme treatment. n = 16, paired t test. b , c Flow cytometry detection and quantification of stromal cells (CD45 – PDPN + ) upon mechanical disaggregation with or without Liberase™ TL proteolytic enzyme treatment. d Flow cytometry of dissociated synovial cells treated with panel of proteolytic Liberase™ TL formulations. Cells gated for viability and CD3 T-cell receptor subunit. Plots representative of n = 4 biologic replicates. e Total RNA yield from synovial tissue dissociated with three concentrations of Liberase™ TL. Two or three technical replicates from nine tissues used for each enzyme concentration. ANOVA with Tukey's comparison, * p
    Figure Legend Snippet: High synovial cell yield, preserved surface markers, and reproducible transcriptomic results with mechanical and enzymatic disaggregation protocol. a Total cell counts per gram from synovial tissue mechanically disaggregated with or without Liberase™ TL proteolytic enzyme treatment. n = 16, paired t test. b , c Flow cytometry detection and quantification of stromal cells (CD45 – PDPN + ) upon mechanical disaggregation with or without Liberase™ TL proteolytic enzyme treatment. d Flow cytometry of dissociated synovial cells treated with panel of proteolytic Liberase™ TL formulations. Cells gated for viability and CD3 T-cell receptor subunit. Plots representative of n = 4 biologic replicates. e Total RNA yield from synovial tissue dissociated with three concentrations of Liberase™ TL. Two or three technical replicates from nine tissues used for each enzyme concentration. ANOVA with Tukey's comparison, * p

    Techniques Used: Flow Cytometry, Cytometry, Concentration Assay

    30) Product Images from "Wheat germ agglutinin–conjugated fluorescent pH sensors for visualizing proton fluxes"

    Article Title: Wheat germ agglutinin–conjugated fluorescent pH sensors for visualizing proton fluxes

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.201912498

    Structured light microscopy of lactate-stimulated proton transport in rat ventricular myocytes labeled with WGA-pHRho. (A) A ∼750-nm illuminated z -plane of a WGA-pHRho–labeled ventricular myocyte ∼2 μm above the coverslip focal plane. (B) Time course of 10 mM lactate perfusion before (red) and after addition of 50 nM AR-C155858 (gray). Plotted data are mean + SEM (shading) for clarity ( n = 6 cells); data were collected at 1 Hz. (C) Pseudocolor image depicting the eroding pixel (5 px) analysis to generate the four annuli. (D) Changes of ΔF/F 0 before, during, and after perfusion of 10 mM lactate at each annulus (A0–A3). (E) Pixel intensity histogram of the four annuli (area normalized) for cell shown in A. (F) Average maximal |ΔF/F 0 | upon wash in (solid bars) and wash out (open bars) of 10 mM lactate for nine cells. One-way ANOVA (Bonferroni's multiple comparison test) was used to determine significance (*, P
    Figure Legend Snippet: Structured light microscopy of lactate-stimulated proton transport in rat ventricular myocytes labeled with WGA-pHRho. (A) A ∼750-nm illuminated z -plane of a WGA-pHRho–labeled ventricular myocyte ∼2 μm above the coverslip focal plane. (B) Time course of 10 mM lactate perfusion before (red) and after addition of 50 nM AR-C155858 (gray). Plotted data are mean + SEM (shading) for clarity ( n = 6 cells); data were collected at 1 Hz. (C) Pseudocolor image depicting the eroding pixel (5 px) analysis to generate the four annuli. (D) Changes of ΔF/F 0 before, during, and after perfusion of 10 mM lactate at each annulus (A0–A3). (E) Pixel intensity histogram of the four annuli (area normalized) for cell shown in A. (F) Average maximal |ΔF/F 0 | upon wash in (solid bars) and wash out (open bars) of 10 mM lactate for nine cells. One-way ANOVA (Bonferroni's multiple comparison test) was used to determine significance (*, P

    Techniques Used: Light Microscopy, Labeling, Whole Genome Amplification

    31) Product Images from "Optimization of Liposomes for Antigen Targeting to Splenic CD169+ Macrophages"

    Article Title: Optimization of Liposomes for Antigen Targeting to Splenic CD169+ Macrophages

    Journal: Pharmaceutics

    doi: 10.3390/pharmaceutics12121138

    Liposomal incorporation of PEG abolishes the interaction between GM3 and CD169. ( A ) Liposomes were coated overnight on an ELISA plate, and binding to WT or mutant CD169 Fc was quantified. Indicated is the average OD450 of 3 independent experiments. ( B – D ) DiD-containing liposomes were incubated with TSn at 4 or 37 °C for 45 min. Representative dot plots following incubation at 37 °C (20 µM liposomes) ( B ). Indicated is the average GMFI ± SD of technical triplicates after incubation at 4 °C (C) and at 37 °C ( D ) (pattern representative of 4 independent experiments). ( E ) DiD-containing liposomes were incubated for 45 min at 37 °C with freshly isolated splenocytes from C57BL/6 WT or W2QR97A-mutant CD169 mice. Subsequently, DiD fluorescence was quantified for distinct cell populations using flow cytometry. Indicated is the GMFI ± SD ( n = 5) (representative of 2 independent experiments). ( F ) DiD-containing liposomes (90 nmol of phospholipid), supplemented with adjuvant, were injected IV in mice, and after 2 h, splenic cell populations were analyzed for DiD fluorescence. Indicated is the average GMFI ± SD ( n = 4). *** p
    Figure Legend Snippet: Liposomal incorporation of PEG abolishes the interaction between GM3 and CD169. ( A ) Liposomes were coated overnight on an ELISA plate, and binding to WT or mutant CD169 Fc was quantified. Indicated is the average OD450 of 3 independent experiments. ( B – D ) DiD-containing liposomes were incubated with TSn at 4 or 37 °C for 45 min. Representative dot plots following incubation at 37 °C (20 µM liposomes) ( B ). Indicated is the average GMFI ± SD of technical triplicates after incubation at 4 °C (C) and at 37 °C ( D ) (pattern representative of 4 independent experiments). ( E ) DiD-containing liposomes were incubated for 45 min at 37 °C with freshly isolated splenocytes from C57BL/6 WT or W2QR97A-mutant CD169 mice. Subsequently, DiD fluorescence was quantified for distinct cell populations using flow cytometry. Indicated is the GMFI ± SD ( n = 5) (representative of 2 independent experiments). ( F ) DiD-containing liposomes (90 nmol of phospholipid), supplemented with adjuvant, were injected IV in mice, and after 2 h, splenic cell populations were analyzed for DiD fluorescence. Indicated is the average GMFI ± SD ( n = 4). *** p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay, Mutagenesis, Incubation, Isolation, Mouse Assay, Fluorescence, Flow Cytometry, Injection

    Inclusion of GM3 in liposomes results in specific binding to CD169-expressing cells in vitro. ( A ) Liposomes were coated overnight on an ELISA plate, and binding to wild-type (WT) or mutant mouse CD169 Fc was quantified. Indicated is the average OD450 of 3 independent experiments. ( B – D ) 1′-dioctadecyl-3,3,3′,3′-tetramethyl indodicarbocyanine (DiD)-containing liposomes were incubated with TSn at 4 or 37 °C for 45 min. Representative dot plots following incubation on 37 °C (20 µM liposomes) ( B ). Indicated is the average geometric mean fluorescence intensity GMFI ± SD of a technical triplicate after incubation at 4 °C ( C ) and at 37 °C ( D ) (pattern representative of 4 independent experiments). ( E ) DiD-containing liposomes were incubated for 45 min at 37 °C with freshly isolated splenocytes from C57BL/6 WT or W2QR97A-mutant CD169 mice. Subsequently, DiD fluorescence was quantified for distinct cell populations using flow cytometry. Indicated is the GMFI ± SD ( n = 5) (representative of 2 independent experiments). * p
    Figure Legend Snippet: Inclusion of GM3 in liposomes results in specific binding to CD169-expressing cells in vitro. ( A ) Liposomes were coated overnight on an ELISA plate, and binding to wild-type (WT) or mutant mouse CD169 Fc was quantified. Indicated is the average OD450 of 3 independent experiments. ( B – D ) 1′-dioctadecyl-3,3,3′,3′-tetramethyl indodicarbocyanine (DiD)-containing liposomes were incubated with TSn at 4 or 37 °C for 45 min. Representative dot plots following incubation on 37 °C (20 µM liposomes) ( B ). Indicated is the average geometric mean fluorescence intensity GMFI ± SD of a technical triplicate after incubation at 4 °C ( C ) and at 37 °C ( D ) (pattern representative of 4 independent experiments). ( E ) DiD-containing liposomes were incubated for 45 min at 37 °C with freshly isolated splenocytes from C57BL/6 WT or W2QR97A-mutant CD169 mice. Subsequently, DiD fluorescence was quantified for distinct cell populations using flow cytometry. Indicated is the GMFI ± SD ( n = 5) (representative of 2 independent experiments). * p

    Techniques Used: Binding Assay, Expressing, In Vitro, Enzyme-linked Immunosorbent Assay, Mutagenesis, Incubation, Fluorescence, Isolation, Mouse Assay, Flow Cytometry

    32) Product Images from "A metabolic interplay coordinated by HLX regulates myeloid differentiation and AML through partly overlapping pathways"

    Article Title: A metabolic interplay coordinated by HLX regulates myeloid differentiation and AML through partly overlapping pathways

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05311-4

    hlx1 regulates hematopoietic stem cell formation and myeloid cell maturation in zebrafish. a – b Whole-mount in situ hybridization (WISH) for runx1 ( a ) and rag1 ( b ) in control or fli: h HLX OE zebrafish embryos at 36 or 96 hpf, respectively. Arrows indicate HSPCs. Numbers in the bottom right corner of panels indicate the number of zebrafish embryos with the indicated phenotype compared to the total number of zebrafish analyzed. Quantification of WISH was performed using FIJI software and statistical significance of three independent experiments in 12 zebrafish embryos was evaluated by Student’s t -test, * P
    Figure Legend Snippet: hlx1 regulates hematopoietic stem cell formation and myeloid cell maturation in zebrafish. a – b Whole-mount in situ hybridization (WISH) for runx1 ( a ) and rag1 ( b ) in control or fli: h HLX OE zebrafish embryos at 36 or 96 hpf, respectively. Arrows indicate HSPCs. Numbers in the bottom right corner of panels indicate the number of zebrafish embryos with the indicated phenotype compared to the total number of zebrafish analyzed. Quantification of WISH was performed using FIJI software and statistical significance of three independent experiments in 12 zebrafish embryos was evaluated by Student’s t -test, * P

    Techniques Used: In Situ Hybridization, Software

    hlx1 regulates the transcription of metabolic genes. a Heatmap of z-transformed normalized gene expression values from RNA-Seq performed on sorted endothelial/hematopoietic ( fli:kaede + ) cells from fli :h HLX OE embryos or endothelial cells ( kdrl:GFP + ) from hlx1 MO at 48 hpf after unsupervised hierarchical clustering with Euclidean distance metric (see also Supplementary Data 1 ). b IPA analysis of deregulated genes in the RNA-Seq data from fli :h HLX OE or hlx1MO embryos at 48 hpf (NBT, P ≤ 0.05 and ≥2 fold change) (also see Supplementary Data 1 ). c Mean density plots of read distribution of lost and gained ATAC-Seq peaks between control and hlx1 MO (see also Supplementary Data 2 ). d Digital genomic footprinting analysis showing average normalized Tn5 insertion profiles around footprinted motifs in merged ATAC peaks as indicated for control and hlx1MO (co-occurrence enrichment computation, z = 3.583 and z = 13.241, respectively). Insertions on the forward and reverse strands are indicated in red and blue, respectively. The numbers of motifs are indicated at the bottom of each panel. e Representative gene tracks from ATAC-Seq data of ETC and ppardb genes. f Heatmap of ETC gene expression in hlx1MO or fli :h HLX OE compared to control from the RNA-Seq analysis. All represented genes show differential chromatin accessibility in ATAC-Seq. g – i qPCR analysis of selected ppar genes in g endothelial cells from hlx1 MO (data representative of two independant experiments, mean + s.d.) or ( h ) whole fli :h HLX OE embryos at 48 hpf ( n = 3, mean + s.d., Student’s t -test, * P
    Figure Legend Snippet: hlx1 regulates the transcription of metabolic genes. a Heatmap of z-transformed normalized gene expression values from RNA-Seq performed on sorted endothelial/hematopoietic ( fli:kaede + ) cells from fli :h HLX OE embryos or endothelial cells ( kdrl:GFP + ) from hlx1 MO at 48 hpf after unsupervised hierarchical clustering with Euclidean distance metric (see also Supplementary Data 1 ). b IPA analysis of deregulated genes in the RNA-Seq data from fli :h HLX OE or hlx1MO embryos at 48 hpf (NBT, P ≤ 0.05 and ≥2 fold change) (also see Supplementary Data 1 ). c Mean density plots of read distribution of lost and gained ATAC-Seq peaks between control and hlx1 MO (see also Supplementary Data 2 ). d Digital genomic footprinting analysis showing average normalized Tn5 insertion profiles around footprinted motifs in merged ATAC peaks as indicated for control and hlx1MO (co-occurrence enrichment computation, z = 3.583 and z = 13.241, respectively). Insertions on the forward and reverse strands are indicated in red and blue, respectively. The numbers of motifs are indicated at the bottom of each panel. e Representative gene tracks from ATAC-Seq data of ETC and ppardb genes. f Heatmap of ETC gene expression in hlx1MO or fli :h HLX OE compared to control from the RNA-Seq analysis. All represented genes show differential chromatin accessibility in ATAC-Seq. g – i qPCR analysis of selected ppar genes in g endothelial cells from hlx1 MO (data representative of two independant experiments, mean + s.d.) or ( h ) whole fli :h HLX OE embryos at 48 hpf ( n = 3, mean + s.d., Student’s t -test, * P

    Techniques Used: Transformation Assay, Expressing, RNA Sequencing Assay, Indirect Immunoperoxidase Assay, Footprinting, Real-time Polymerase Chain Reaction

    PPARδ modulation can rescue HSPC formation and myeloid differentiation in zebrafish. a Oxygen consumption rate (OCR) in control or fli :h HLX OE zebrafish. Representative plot from three independent experiments (mean ± s.d.). b – c Mitochondrial membrane potential measured by TMRM stain (left panel) and mitochondrial/nuclear DNA content analysis (right panel) in ( b ) control and fli :h HLX OE embryos or in ( c ) endothelial/hematopoietic cells ( fli: GFP positive) of control and hlx1MO embryos at 48 hpf ( n = 3, mean + s.d., Student’s t -test, * P
    Figure Legend Snippet: PPARδ modulation can rescue HSPC formation and myeloid differentiation in zebrafish. a Oxygen consumption rate (OCR) in control or fli :h HLX OE zebrafish. Representative plot from three independent experiments (mean ± s.d.). b – c Mitochondrial membrane potential measured by TMRM stain (left panel) and mitochondrial/nuclear DNA content analysis (right panel) in ( b ) control and fli :h HLX OE embryos or in ( c ) endothelial/hematopoietic cells ( fli: GFP positive) of control and hlx1MO embryos at 48 hpf ( n = 3, mean + s.d., Student’s t -test, * P

    Techniques Used: Staining, Positive Control

    33) Product Images from "MHC class II expression and potential antigen-presenting cells in the retina during experimental autoimmune uveitis"

    Article Title: MHC class II expression and potential antigen-presenting cells in the retina during experimental autoimmune uveitis

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-017-0915-5

    MHC class II expression correlates to EAU severity. Three weeks after adoptive transfer, retinas were carefully dissected, cut into small pieces, and dissociated by incubation with Liberase DL and DNase I at 37 °C for 45 min. Naive eyes were used as control. The single-cell suspensions, excluding dead cells (DAPI+) were analyzed by flow cytometry for MHC II expression using fluorochrome-conjugated specific antibodies. For Figures B to E, data are from 1 representative mouse out of 21 independent mice (5 control, 8 low-score EAU and 8 high-score EAU mice). a Number of MHC class II-expressing cells (normalized per 1 million analyzed retinal cells) for different EAU clinical scores. Data represented: mean ± SEM, ANOVA, and Tukey post-hoc multiple comparisons test, ** p
    Figure Legend Snippet: MHC class II expression correlates to EAU severity. Three weeks after adoptive transfer, retinas were carefully dissected, cut into small pieces, and dissociated by incubation with Liberase DL and DNase I at 37 °C for 45 min. Naive eyes were used as control. The single-cell suspensions, excluding dead cells (DAPI+) were analyzed by flow cytometry for MHC II expression using fluorochrome-conjugated specific antibodies. For Figures B to E, data are from 1 representative mouse out of 21 independent mice (5 control, 8 low-score EAU and 8 high-score EAU mice). a Number of MHC class II-expressing cells (normalized per 1 million analyzed retinal cells) for different EAU clinical scores. Data represented: mean ± SEM, ANOVA, and Tukey post-hoc multiple comparisons test, ** p

    Techniques Used: Expressing, Adoptive Transfer Assay, Incubation, Flow Cytometry, Cytometry, Mouse Assay

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