anti cd45 pe  (Thermo Fisher)


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    Structured Review

    Thermo Fisher anti cd45 pe
    LILRB2 blockade reprograms lung TAM maturation in vitro and in vivo. ( A ) FACS gating analysis of primary human CD33 + monocytes (1 × 10 5 cells per well) with 5 days of coculture of A549 cells (1 × 10 3 cells per well). ( B ) CD14, CD16, CD163, and DC-SIGN expression among live CD33 + CD14 + myeloid cells from A . IgG-treated cells (black line) are overlaid by anti-LILRB2–treated cells (αLILRB2, red line). ( C ) FACS gating strategy for identifying human <t>CD45</t> + CD33 + macrophages from NSG-SGM3 immunodeficient mice inoculated s.c. with A549 tumor and CD33 + monocytes 12 days earlier. ( D . Data were averaged from 4–8 tumor samples per group per donor, 2–4 mice per group per donor, 3 independent experiments. * P
    Anti Cd45 Pe, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Blocking immunoinhibitory receptor LILRB2 reprograms tumor-associated myeloid cells and promotes antitumor immunity"

    Article Title: Blocking immunoinhibitory receptor LILRB2 reprograms tumor-associated myeloid cells and promotes antitumor immunity

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI97570

    LILRB2 blockade reprograms lung TAM maturation in vitro and in vivo. ( A ) FACS gating analysis of primary human CD33 + monocytes (1 × 10 5 cells per well) with 5 days of coculture of A549 cells (1 × 10 3 cells per well). ( B ) CD14, CD16, CD163, and DC-SIGN expression among live CD33 + CD14 + myeloid cells from A . IgG-treated cells (black line) are overlaid by anti-LILRB2–treated cells (αLILRB2, red line). ( C ) FACS gating strategy for identifying human CD45 + CD33 + macrophages from NSG-SGM3 immunodeficient mice inoculated s.c. with A549 tumor and CD33 + monocytes 12 days earlier. ( D . Data were averaged from 4–8 tumor samples per group per donor, 2–4 mice per group per donor, 3 independent experiments. * P
    Figure Legend Snippet: LILRB2 blockade reprograms lung TAM maturation in vitro and in vivo. ( A ) FACS gating analysis of primary human CD33 + monocytes (1 × 10 5 cells per well) with 5 days of coculture of A549 cells (1 × 10 3 cells per well). ( B ) CD14, CD16, CD163, and DC-SIGN expression among live CD33 + CD14 + myeloid cells from A . IgG-treated cells (black line) are overlaid by anti-LILRB2–treated cells (αLILRB2, red line). ( C ) FACS gating strategy for identifying human CD45 + CD33 + macrophages from NSG-SGM3 immunodeficient mice inoculated s.c. with A549 tumor and CD33 + monocytes 12 days earlier. ( D . Data were averaged from 4–8 tumor samples per group per donor, 2–4 mice per group per donor, 3 independent experiments. * P

    Techniques Used: In Vitro, In Vivo, FACS, Expressing, Mouse Assay

    Tumor-infiltrating myeloid cells derived from lung cancer and mesothelioma patients respond to LILRB2 blockade. ( A ) Characterization of myeloid cell populations isolated from NSCLC excised tumor. Cells were gated on DAPI – CD45 + live leukocyte cells. PMN-MDSC, M-MDSC, DC, and TAM populations were identified. ( B ) Representative LILRB1–LILRB4 staining among CD33 + gate from A . ( C ) LILRB MFI of multiple patient biopsies ( n = 5–6) shown gated on the MDSC (left) and TAM (right) gates. ( D ) Lung cancer– and mesothelioma-derived infiltrating lymphocytes (TILs) were cultured with M-CSF plus IFN-γ with Ig controls or anti-LILRB2 (αLILRB2) for 48 hours. The supernatants were evaluated for TNF-α and IL-10 secretion ( n = 15). *** P
    Figure Legend Snippet: Tumor-infiltrating myeloid cells derived from lung cancer and mesothelioma patients respond to LILRB2 blockade. ( A ) Characterization of myeloid cell populations isolated from NSCLC excised tumor. Cells were gated on DAPI – CD45 + live leukocyte cells. PMN-MDSC, M-MDSC, DC, and TAM populations were identified. ( B ) Representative LILRB1–LILRB4 staining among CD33 + gate from A . ( C ) LILRB MFI of multiple patient biopsies ( n = 5–6) shown gated on the MDSC (left) and TAM (right) gates. ( D ) Lung cancer– and mesothelioma-derived infiltrating lymphocytes (TILs) were cultured with M-CSF plus IFN-γ with Ig controls or anti-LILRB2 (αLILRB2) for 48 hours. The supernatants were evaluated for TNF-α and IL-10 secretion ( n = 15). *** P

    Techniques Used: Derivative Assay, Isolation, Staining, Cell Culture

    2) Product Images from "Age-related susceptibility to grey matter demyelination and neurodegeneration is associated with meningeal neutrophil accumulation in an animal model of Multiple Sclerosis"

    Article Title: Age-related susceptibility to grey matter demyelination and neurodegeneration is associated with meningeal neutrophil accumulation in an animal model of Multiple Sclerosis

    Journal: bioRxiv

    doi: 10.1101/2021.12.23.474008

    Ageing does not impact the frequency of lymphocytes or the ability of T cells to produce cytokines in SJL/J A/T EAE mice at peak disease stage. Whole brains and spinal cords of young and old SJL/J A/T EAE and naive mice were analyzed by flow cytometry. (A-B) Frequencies of B220+CD19+ B cells, CD4+ and CD8+ T cells of all CD45+ cells in the brains and spinal cords of old vs young SJL/J A/T EAE mice. Stats by Mann-Whitney U showed no statistical difference between the groups. Cells were also stimulated with PMA and ionomycin for 5 hours. (C-D) Frequencies and absolute numbers of cytokine-producing CD4+ T cells was assessed using intra-cellular staining for GM-CSF, IFNγ, and IL-17. Statistical test by Mann-Whitney showed no significant differences between the groups. Error bars indicate mean with SD.
    Figure Legend Snippet: Ageing does not impact the frequency of lymphocytes or the ability of T cells to produce cytokines in SJL/J A/T EAE mice at peak disease stage. Whole brains and spinal cords of young and old SJL/J A/T EAE and naive mice were analyzed by flow cytometry. (A-B) Frequencies of B220+CD19+ B cells, CD4+ and CD8+ T cells of all CD45+ cells in the brains and spinal cords of old vs young SJL/J A/T EAE mice. Stats by Mann-Whitney U showed no statistical difference between the groups. Cells were also stimulated with PMA and ionomycin for 5 hours. (C-D) Frequencies and absolute numbers of cytokine-producing CD4+ T cells was assessed using intra-cellular staining for GM-CSF, IFNγ, and IL-17. Statistical test by Mann-Whitney showed no significant differences between the groups. Error bars indicate mean with SD.

    Techniques Used: Mouse Assay, Flow Cytometry, MANN-WHITNEY, Staining

    3) Product Images from "Blocking immunoinhibitory receptor LILRB2 reprograms tumor-associated myeloid cells and promotes antitumor immunity"

    Article Title: Blocking immunoinhibitory receptor LILRB2 reprograms tumor-associated myeloid cells and promotes antitumor immunity

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI97570

    LILRB2 blockade reprograms lung TAM maturation in vitro and in vivo. ( A ) FACS gating analysis of primary human CD33 + monocytes (1 × 10 5 cells per well) with 5 days of coculture of A549 cells (1 × 10 3 cells per well). ( B ) CD14, CD16, CD163, and DC-SIGN expression among live CD33 + CD14 + myeloid cells from A . IgG-treated cells (black line) are overlaid by anti-LILRB2–treated cells (αLILRB2, red line). ( C ) FACS gating strategy for identifying human CD45 + CD33 + macrophages from NSG-SGM3 immunodeficient mice inoculated s.c. with A549 tumor and CD33 + monocytes 12 days earlier. ( D . Data were averaged from 4–8 tumor samples per group per donor, 2–4 mice per group per donor, 3 independent experiments. * P
    Figure Legend Snippet: LILRB2 blockade reprograms lung TAM maturation in vitro and in vivo. ( A ) FACS gating analysis of primary human CD33 + monocytes (1 × 10 5 cells per well) with 5 days of coculture of A549 cells (1 × 10 3 cells per well). ( B ) CD14, CD16, CD163, and DC-SIGN expression among live CD33 + CD14 + myeloid cells from A . IgG-treated cells (black line) are overlaid by anti-LILRB2–treated cells (αLILRB2, red line). ( C ) FACS gating strategy for identifying human CD45 + CD33 + macrophages from NSG-SGM3 immunodeficient mice inoculated s.c. with A549 tumor and CD33 + monocytes 12 days earlier. ( D . Data were averaged from 4–8 tumor samples per group per donor, 2–4 mice per group per donor, 3 independent experiments. * P

    Techniques Used: In Vitro, In Vivo, FACS, Expressing, Mouse Assay

    Tumor-infiltrating myeloid cells derived from lung cancer and mesothelioma patients respond to LILRB2 blockade. ( A ) Characterization of myeloid cell populations isolated from NSCLC excised tumor. Cells were gated on DAPI – CD45 + live leukocyte cells. PMN-MDSC, M-MDSC, DC, and TAM populations were identified. ( B ) Representative LILRB1–LILRB4 staining among CD33 + gate from A . ( C ) LILRB MFI of multiple patient biopsies ( n = 5–6) shown gated on the MDSC (left) and TAM (right) gates. ( D ) Lung cancer– and mesothelioma-derived infiltrating lymphocytes (TILs) were cultured with M-CSF plus IFN-γ with Ig controls or anti-LILRB2 (αLILRB2) for 48 hours. The supernatants were evaluated for TNF-α and IL-10 secretion ( n = 15). *** P
    Figure Legend Snippet: Tumor-infiltrating myeloid cells derived from lung cancer and mesothelioma patients respond to LILRB2 blockade. ( A ) Characterization of myeloid cell populations isolated from NSCLC excised tumor. Cells were gated on DAPI – CD45 + live leukocyte cells. PMN-MDSC, M-MDSC, DC, and TAM populations were identified. ( B ) Representative LILRB1–LILRB4 staining among CD33 + gate from A . ( C ) LILRB MFI of multiple patient biopsies ( n = 5–6) shown gated on the MDSC (left) and TAM (right) gates. ( D ) Lung cancer– and mesothelioma-derived infiltrating lymphocytes (TILs) were cultured with M-CSF plus IFN-γ with Ig controls or anti-LILRB2 (αLILRB2) for 48 hours. The supernatants were evaluated for TNF-α and IL-10 secretion ( n = 15). *** P

    Techniques Used: Derivative Assay, Isolation, Staining, Cell Culture

    4) Product Images from "The human placenta is a hematopoietic organ during the embryonic and fetal periods of development"

    Article Title: The human placenta is a hematopoietic organ during the embryonic and fetal periods of development

    Journal:

    doi: 10.1016/j.ydbio.2008.11.017

    The hematopoietic compartment of the placenta changes during gestation. ( A ) Box plot of total CD34 ++ CD45 low placental cells grouped by gestational age (in weeks). ( B ) Box plot of the number of CD34 ++ CD45 low cells per gram of tissue grouped by gestational
    Figure Legend Snippet: The hematopoietic compartment of the placenta changes during gestation. ( A ) Box plot of total CD34 ++ CD45 low placental cells grouped by gestational age (in weeks). ( B ) Box plot of the number of CD34 ++ CD45 low cells per gram of tissue grouped by gestational

    Techniques Used:

    Hematopoietic progenitors were present in chorionic villi and the chorioamniotic membranes. Freshly isolated light-density cells from villi and fetal membranes ( A , pooled 6.3 + 6.5 weeks; B , 15.3 weeks) were stained with CD34, CD45 and PI and analyzed
    Figure Legend Snippet: Hematopoietic progenitors were present in chorionic villi and the chorioamniotic membranes. Freshly isolated light-density cells from villi and fetal membranes ( A , pooled 6.3 + 6.5 weeks; B , 15.3 weeks) were stained with CD34, CD45 and PI and analyzed

    Techniques Used: Isolation, Staining

    Localization and morphology of CD34 + CD45 + cells within the human placenta. ( A ) Chorionic villi biopsied from a 9-week human placenta embedded in gelatin to demonstrate the macroscopic anatomy of these structures (2x magnification). fs, fetal side; fm,
    Figure Legend Snippet: Localization and morphology of CD34 + CD45 + cells within the human placenta. ( A ) Chorionic villi biopsied from a 9-week human placenta embedded in gelatin to demonstrate the macroscopic anatomy of these structures (2x magnification). fs, fetal side; fm,

    Techniques Used:

    CD34 ++ CD45 + placental cells have multilineage hematopoietic potential. CD34 ++ CD45 low cells sorted from a 19-week placenta were cultured at 7.5 × 10 3 cells/well in 48-well plates in SDM supplemented with SCF+FL+IL-7+IL-15+GM-CSF+IL-3 ( A ) or SCF+FL+Epo+GM-CSF+TPO
    Figure Legend Snippet: CD34 ++ CD45 + placental cells have multilineage hematopoietic potential. CD34 ++ CD45 low cells sorted from a 19-week placenta were cultured at 7.5 × 10 3 cells/well in 48-well plates in SDM supplemented with SCF+FL+IL-7+IL-15+GM-CSF+IL-3 ( A ) or SCF+FL+Epo+GM-CSF+TPO

    Techniques Used: Cell Culture

    5) Product Images from "Anti-inflammatory protein TSG-6 secreted by bone marrow mesenchymal stem cells attenuates neuropathic pain by inhibiting the TLR2/MyD88/NF-κB signaling pathway in spinal microglia"

    Article Title: Anti-inflammatory protein TSG-6 secreted by bone marrow mesenchymal stem cells attenuates neuropathic pain by inhibiting the TLR2/MyD88/NF-κB signaling pathway in spinal microglia

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-020-1731-x

    Characterization of BMSCs. Morphological observation of rat BMSCs ( a ) used in this study. Scale bar: 200 μm. b Flow cytometric analysis of BMSCs surface markers. The cells were positive for CD90, CD29, and CD105, and negative for CD45, CD34, and CD11b. c The multidifferentiation potential of BMSCs in vitro. Alizarin red S staining was used to evaluate osteogenic differentiation, Alcian blue staining was used to evaluate chondrogenic differentiation, and oil Red O staining was used to evaluate adipogenic differentiation. Scale bar: 30 μm
    Figure Legend Snippet: Characterization of BMSCs. Morphological observation of rat BMSCs ( a ) used in this study. Scale bar: 200 μm. b Flow cytometric analysis of BMSCs surface markers. The cells were positive for CD90, CD29, and CD105, and negative for CD45, CD34, and CD11b. c The multidifferentiation potential of BMSCs in vitro. Alizarin red S staining was used to evaluate osteogenic differentiation, Alcian blue staining was used to evaluate chondrogenic differentiation, and oil Red O staining was used to evaluate adipogenic differentiation. Scale bar: 30 μm

    Techniques Used: In Vitro, Staining

    6) Product Images from "Borrelia peptidoglycan interacting Protein (BpiP) contributes to the fitness of Borrelia burgdorferi against host-derived factors and influences virulence in mouse models of Lyme disease"

    Article Title: Borrelia peptidoglycan interacting Protein (BpiP) contributes to the fitness of Borrelia burgdorferi against host-derived factors and influences virulence in mouse models of Lyme disease

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1009535

    Phagocytosis of bpiP mt by activated macrophages and dendritic cells. All three strains (wt, mt and ct) were labeled with CFSE dye as described in the Materials and Methods sections and incubated with activated macrophages or dendritic cells for 10 mins. The infected cells were then harvested and stained for macrophage positive markers CD45 and F4/80 using antibodies conjugated to the fluorophores PE and APC respectively. Macrophages were identified as CD45 + (Ex (max)/ Em (max) : 565/578; channel 3) /F480 + (Ex (max)/ Em (max) : 652/660; channel 11) cells while DC were identified as CD45 + /CD11c + (conjugated to the flurophore BV421, Ex (max)/ Em (max) : 495/520; channel 7) but F480 negative cells. The samples were analyzed by Image Flow Cytometry with (A) macrophage markers detected using channels 3 and 11 corresponding to excitation wavelengths of 488nm and 642 nm, respectively and (C) DC markers were detected using channel 7 with an excitation wavelength of 405nm. CFSE-labeled Bb was detected using channel 2 ( Bb /CFSE Ex (max)/ Em (max) : 495/520) with an excitation wavelength of 488nm. Data shown are means of three technical replicates with error bars representing Standard Error Mean. Pictures are representative of data obtained from one of three independent experiments. The percentage of internalized Bb within (B) macrophages and (D) dendritic cells was quantified and analyzed statistically using unpaired t test. The asterisks indicate levels of significance as follows: **, p
    Figure Legend Snippet: Phagocytosis of bpiP mt by activated macrophages and dendritic cells. All three strains (wt, mt and ct) were labeled with CFSE dye as described in the Materials and Methods sections and incubated with activated macrophages or dendritic cells for 10 mins. The infected cells were then harvested and stained for macrophage positive markers CD45 and F4/80 using antibodies conjugated to the fluorophores PE and APC respectively. Macrophages were identified as CD45 + (Ex (max)/ Em (max) : 565/578; channel 3) /F480 + (Ex (max)/ Em (max) : 652/660; channel 11) cells while DC were identified as CD45 + /CD11c + (conjugated to the flurophore BV421, Ex (max)/ Em (max) : 495/520; channel 7) but F480 negative cells. The samples were analyzed by Image Flow Cytometry with (A) macrophage markers detected using channels 3 and 11 corresponding to excitation wavelengths of 488nm and 642 nm, respectively and (C) DC markers were detected using channel 7 with an excitation wavelength of 405nm. CFSE-labeled Bb was detected using channel 2 ( Bb /CFSE Ex (max)/ Em (max) : 495/520) with an excitation wavelength of 488nm. Data shown are means of three technical replicates with error bars representing Standard Error Mean. Pictures are representative of data obtained from one of three independent experiments. The percentage of internalized Bb within (B) macrophages and (D) dendritic cells was quantified and analyzed statistically using unpaired t test. The asterisks indicate levels of significance as follows: **, p

    Techniques Used: Labeling, Incubation, Infection, Staining, Flow Cytometry

    7) Product Images from "Novel immunotherapeutic effects of topically administered ripasudil (K-115) on corneal allograft survival"

    Article Title: Novel immunotherapeutic effects of topically administered ripasudil (K-115) on corneal allograft survival

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-76882-w

    Leukocyte infiltration and inflammation-related mRNA levels in corneal grafts. ( A ) Representative flow cytometry plots and ( B ) statistical analysis of flow cytometry data showing the frequency of occurrence of infiltrating CD45 + leukocytes in grafted corneas at day 14 post-transplantation (n = 3, one-way ANOVA; * p
    Figure Legend Snippet: Leukocyte infiltration and inflammation-related mRNA levels in corneal grafts. ( A ) Representative flow cytometry plots and ( B ) statistical analysis of flow cytometry data showing the frequency of occurrence of infiltrating CD45 + leukocytes in grafted corneas at day 14 post-transplantation (n = 3, one-way ANOVA; * p

    Techniques Used: Flow Cytometry, Transplantation Assay

    8) Product Images from "Microglia Require CD4 T Cells to Complete the Fetal-to-Adult Transition"

    Article Title: Microglia Require CD4 T Cells to Complete the Fetal-to-Adult Transition

    Journal: Cell

    doi: 10.1016/j.cell.2020.06.026

    Brain Conventional CD4 T Cells Are Expanded by Exposure to the Microbiome (A and B) Perfused mouse brains were compared to blood by high-dimensional flow cytometry from gnotobiotic, SPF, and dirty co-housed mice (n = 6,10,12). CD4 T cells in perfused brain as (A) a percentage of CD45 + cells and (B) absolute numbers of CD4 T cells in the brain. (C and D) t-SNE of conventional (C) T cells and (D) Tregs built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, and CTLA4, with quantified FlowSOM clusters. p values represent cross-entropy comparison to SPF mice. See also Figure S5 .
    Figure Legend Snippet: Brain Conventional CD4 T Cells Are Expanded by Exposure to the Microbiome (A and B) Perfused mouse brains were compared to blood by high-dimensional flow cytometry from gnotobiotic, SPF, and dirty co-housed mice (n = 6,10,12). CD4 T cells in perfused brain as (A) a percentage of CD45 + cells and (B) absolute numbers of CD4 T cells in the brain. (C and D) t-SNE of conventional (C) T cells and (D) Tregs built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, and CTLA4, with quantified FlowSOM clusters. p values represent cross-entropy comparison to SPF mice. See also Figure S5 .

    Techniques Used: Flow Cytometry, Mouse Assay

    Phenotypic Profile of Brain-Resident TCR Transgenic CD4 T Cells, Related to Figure 4 CD4 T cells from perfused brains of TCR transgenic mice and wild-type controls (n = 5) were assessed by high parameter flow cytometry for CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4. (A) tSNE of CD4 + Foxp3 - conventional 2D2 T cells, gated on CD4 + Foxp3 - CD3 + CD8 - CD45 + cells and subdivided into transgene-expressing (Vα3.2 + Vβ11 + ) and transgene-non-expressing (Vα3.2 - Vβ11 - ) cells. The tSNE was built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4. FlowSOM clusters are illustrated in color and (B) quantified. (C) Heatmap of expression changes between transgene-expressing (Vα3.2 + Vβ11 + ) and transgene-non-expressing (Vα3.2 - Vβ11 - ) conventional 2D2 CD4 + T cells. (D) tSNE of CD4 + Foxp3 + 2D2 regulatory T cells, gated on CD4 + Foxp3 + CD3 + CD8 - CD45 + cells and subdivided into transgene-expressing (Vα3.2 + Vβ11 + ) and transgene-non-expressing (Vα3.2 - Vβ11 - ) cells. The tSNE was built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4. FlowSOM clusters are illustrated in color and (E) quantified. (F) Heatmap of expression changes between transgene-expressing (Vα2 + Vβ5 + ) and transgene-non-expressing (Vα2 - Vβ5 - ) regulatory T cells. (G) tSNE of CD4 + Foxp3 - conventional OT-II T cells, gated on CD4 + Foxp3 - CD3 + CD8 - CD45 + cells and subdivided into transgene-expressing (Vα2 + Vβ5 + ) and transgene-non-expressing (Vα2 - Vβ5 - ) cells. The tSNE was built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4. FlowSOM clusters are illustrated in color and (H) quantified. (I) Heatmap of expression changes between transgene-expressing (Vα2 + Vβ5 + ) and transgene-non-expressing (Vα2 - Vβ5 - ) conventional OT-II CD4 + T cells.
    Figure Legend Snippet: Phenotypic Profile of Brain-Resident TCR Transgenic CD4 T Cells, Related to Figure 4 CD4 T cells from perfused brains of TCR transgenic mice and wild-type controls (n = 5) were assessed by high parameter flow cytometry for CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4. (A) tSNE of CD4 + Foxp3 - conventional 2D2 T cells, gated on CD4 + Foxp3 - CD3 + CD8 - CD45 + cells and subdivided into transgene-expressing (Vα3.2 + Vβ11 + ) and transgene-non-expressing (Vα3.2 - Vβ11 - ) cells. The tSNE was built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4. FlowSOM clusters are illustrated in color and (B) quantified. (C) Heatmap of expression changes between transgene-expressing (Vα3.2 + Vβ11 + ) and transgene-non-expressing (Vα3.2 - Vβ11 - ) conventional 2D2 CD4 + T cells. (D) tSNE of CD4 + Foxp3 + 2D2 regulatory T cells, gated on CD4 + Foxp3 + CD3 + CD8 - CD45 + cells and subdivided into transgene-expressing (Vα3.2 + Vβ11 + ) and transgene-non-expressing (Vα3.2 - Vβ11 - ) cells. The tSNE was built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4. FlowSOM clusters are illustrated in color and (E) quantified. (F) Heatmap of expression changes between transgene-expressing (Vα2 + Vβ5 + ) and transgene-non-expressing (Vα2 - Vβ5 - ) regulatory T cells. (G) tSNE of CD4 + Foxp3 - conventional OT-II T cells, gated on CD4 + Foxp3 - CD3 + CD8 - CD45 + cells and subdivided into transgene-expressing (Vα2 + Vβ5 + ) and transgene-non-expressing (Vα2 - Vβ5 - ) cells. The tSNE was built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4. FlowSOM clusters are illustrated in color and (H) quantified. (I) Heatmap of expression changes between transgene-expressing (Vα2 + Vβ5 + ) and transgene-non-expressing (Vα2 - Vβ5 - ) conventional OT-II CD4 + T cells.

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

    Brain T Cells Alter Gene Expression in Microglia, Related to Figure 6 (A) Graph of CD4 + T cells in wild-type and MHC II KO mice in the spleen and perfused brain, as a percentage of CD45 + cells (n = 4). (B) Single cell RNaseq data from CD11b + cells purified from wild-type mice and MHC II-deficient mice were assessed for gene-expression. tSNE dimensionality reduction is used for cluster display, with lineage marker expression indicated by color for Cx3cr1 , P2ry12 , Tmem119 , ApoE , S100a9 , Mrc1 , Cd74 and S100a4 . (C) Gene expression from isolated microglia from adult male wild-type and MHC class II KO mice (n = 4,4) were measured by qPCR. The expression of the indicated genes, selected from the differentially-expressed single cell sequencing dataset ( Ccr6 upregulated in MHC II KO microglia, other genes downregulated in MHC II KO microglia) was calculated relative to Ppia rRNA. P value s were obtained by two-tailed Mann-Whitney U test. (D) Single cell RNaseq data was generated on microglia collected from wild-type mice and MHC II-deficient mice. Volcano plots of wild-type versus MHC II-deficient clusters 1, cluster 2, cluster 3, cluster 4, cluster 5, cluster 6, cluster 7 and cluster 8 microglia, perivascular macrophages, macrophages, monocytes and granulocytes. Labeled genes with a differential expression of more than 1-log fold change (p
    Figure Legend Snippet: Brain T Cells Alter Gene Expression in Microglia, Related to Figure 6 (A) Graph of CD4 + T cells in wild-type and MHC II KO mice in the spleen and perfused brain, as a percentage of CD45 + cells (n = 4). (B) Single cell RNaseq data from CD11b + cells purified from wild-type mice and MHC II-deficient mice were assessed for gene-expression. tSNE dimensionality reduction is used for cluster display, with lineage marker expression indicated by color for Cx3cr1 , P2ry12 , Tmem119 , ApoE , S100a9 , Mrc1 , Cd74 and S100a4 . (C) Gene expression from isolated microglia from adult male wild-type and MHC class II KO mice (n = 4,4) were measured by qPCR. The expression of the indicated genes, selected from the differentially-expressed single cell sequencing dataset ( Ccr6 upregulated in MHC II KO microglia, other genes downregulated in MHC II KO microglia) was calculated relative to Ppia rRNA. P value s were obtained by two-tailed Mann-Whitney U test. (D) Single cell RNaseq data was generated on microglia collected from wild-type mice and MHC II-deficient mice. Volcano plots of wild-type versus MHC II-deficient clusters 1, cluster 2, cluster 3, cluster 4, cluster 5, cluster 6, cluster 7 and cluster 8 microglia, perivascular macrophages, macrophages, monocytes and granulocytes. Labeled genes with a differential expression of more than 1-log fold change (p

    Techniques Used: Expressing, Mouse Assay, Purification, Marker, Isolation, Real-time Polymerase Chain Reaction, Sequencing, Two Tailed Test, MANN-WHITNEY, Generated, Labeling

    Microglial Density Morphology Diverges during Development between Wild-Type and MHC II-Deficient Mice, Related to Figure 7 Microglia structure and morphology were assessed in the brain of wild-type and MHC II-deficient mice at post-natal day 0 (striatum), day 7 and 15 weeks of age. (A) Representative 20 × view of confocal images of Iba1 immunostaining showing microglial density; scale = 50μm. (B) Quantification of microglia density at post-natal day 0, day 7 and 15 weeks (n = 4,2,2,5,4,4). (C) Representative 20 × view of confocal images Iba1 labeling (red) from the post-natal day 0 (striatum) and day 7 (cortex). Scale = 50μm, arrows indicate phagocytic microglia containing engulfed DAPI + nucleus. (D) Quantification of microglia exhibiting phagocytotic buds (n = 4,2,2,5). (E) Representative 40 × images showing microglia morphology, process extensions and ramification at 15 weeks; scale = 50μm. (F) Quantification of the proportion of microglia with a maximum enclosing radius out to varying distances. Number of microglia analyzed: wild-type = 37; MHC II-deficient mice = 44 (n = 4,4). (G) Quantification of the total number of process intersections in microglia. (H) Sholl analysis of microglia process intersections per radii (spaced with the interval of 10 μm) from the soma. Dots represent each microglia, n = 8-10 microglia/mouse (n = 4,4). Fligner-Killeen non-parametric test for difference in variance. (I) MHC II-deficient mice and wild-type siblings were assessed for behavioral abnormalities. Time spent on the rod, average of 4 repeated tests of 300 s (n = 24,25). (J) Sociability test trials to monitor the interaction with a stranger mouse (S1) compared to a empty chamber (E1) (K) and the social preference for a new stranger (S2), with interaction with repeated stranger (S1) and new stranger (S2). (n = 24,23). (L) Freezing behavior over time during context acquisition conditioning (n = 13,14). (M) Marble burying test (n = 9,14). (N) Wild-type mice were housed under standard SPF conditions, or placed under behavioral modification in the form of isolated or environmental enrichment (n = 18, 15, 10). Mice were compared by high parameter flow cytometry of the blood and perfused brain. CD4 T cells as absolute numbers of conventional cells and Tregs in the brain. P value refers to comparison of conventional T cells. (O) Proportion of Foxp3 + cells within the CD4 T cell population in blood and brain. (P) tSNE of CD4 + Foxp3 - T cells gated on CD4 + Foxp3 - CD3 + CD8 - CD45 + cells or (Q) CD4 + Foxp3 + CD3 + CD8 - CD45 + cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4. P values represent cross-entropy comparison to control mice. FlowSOM clusters are illustrated in color. (R) Proportion of naive (top) and activated (bottom) cells within the conventional and (S) Treg populations in the blood and brain. Mean ± SEM.
    Figure Legend Snippet: Microglial Density Morphology Diverges during Development between Wild-Type and MHC II-Deficient Mice, Related to Figure 7 Microglia structure and morphology were assessed in the brain of wild-type and MHC II-deficient mice at post-natal day 0 (striatum), day 7 and 15 weeks of age. (A) Representative 20 × view of confocal images of Iba1 immunostaining showing microglial density; scale = 50μm. (B) Quantification of microglia density at post-natal day 0, day 7 and 15 weeks (n = 4,2,2,5,4,4). (C) Representative 20 × view of confocal images Iba1 labeling (red) from the post-natal day 0 (striatum) and day 7 (cortex). Scale = 50μm, arrows indicate phagocytic microglia containing engulfed DAPI + nucleus. (D) Quantification of microglia exhibiting phagocytotic buds (n = 4,2,2,5). (E) Representative 40 × images showing microglia morphology, process extensions and ramification at 15 weeks; scale = 50μm. (F) Quantification of the proportion of microglia with a maximum enclosing radius out to varying distances. Number of microglia analyzed: wild-type = 37; MHC II-deficient mice = 44 (n = 4,4). (G) Quantification of the total number of process intersections in microglia. (H) Sholl analysis of microglia process intersections per radii (spaced with the interval of 10 μm) from the soma. Dots represent each microglia, n = 8-10 microglia/mouse (n = 4,4). Fligner-Killeen non-parametric test for difference in variance. (I) MHC II-deficient mice and wild-type siblings were assessed for behavioral abnormalities. Time spent on the rod, average of 4 repeated tests of 300 s (n = 24,25). (J) Sociability test trials to monitor the interaction with a stranger mouse (S1) compared to a empty chamber (E1) (K) and the social preference for a new stranger (S2), with interaction with repeated stranger (S1) and new stranger (S2). (n = 24,23). (L) Freezing behavior over time during context acquisition conditioning (n = 13,14). (M) Marble burying test (n = 9,14). (N) Wild-type mice were housed under standard SPF conditions, or placed under behavioral modification in the form of isolated or environmental enrichment (n = 18, 15, 10). Mice were compared by high parameter flow cytometry of the blood and perfused brain. CD4 T cells as absolute numbers of conventional cells and Tregs in the brain. P value refers to comparison of conventional T cells. (O) Proportion of Foxp3 + cells within the CD4 T cell population in blood and brain. (P) tSNE of CD4 + Foxp3 - T cells gated on CD4 + Foxp3 - CD3 + CD8 - CD45 + cells or (Q) CD4 + Foxp3 + CD3 + CD8 - CD45 + cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4. P values represent cross-entropy comparison to control mice. FlowSOM clusters are illustrated in color. (R) Proportion of naive (top) and activated (bottom) cells within the conventional and (S) Treg populations in the blood and brain. Mean ± SEM.

    Techniques Used: Mouse Assay, Immunostaining, Labeling, Modification, Isolation, Flow Cytometry

    Brain-Resident CD4 T Cells Acquire a Residency Phenotype In Situ during a Prolonged Brain Transit (A) Schematic of parabiosis experiments (n = 12,12,18,16,14). (B and C) Curve of best fit for the origin of conventional (B) T cells or (C) Tregs showing CD69 − and CD69 + in the blood and brain. (D) Derived median dwell times. (E and F) t-SNE of conventional (E) T cells and (F) Tregs built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, and CTLA4. FlowSOM clusters represented in color. Host and incoming cells were defined on CD45.1 versus CD45.2 expression, and are shown at the 2-, 4-, and 8-week time points. (G and H) CD69 histograms for CD4 conventional (G) T cells and (H) Tregs. (I and J) Population flow diagrams for conventional (I) T cells and (J) Tregs, in homeostatic state. Circle areas represent population frequencies, calculated independently for blood and brain. Small black circles represent cell death. The size of arrow ends is proportional to the rate of population flow. Numbers display the corresponding entry or exit rate, in events/1,000 cells/day. Numbers with asterisk denote rates with high estimation uncertainty. Population transitions with rates lower than 0.1/1,000 cells/day are not shown. See also Figure S3 .
    Figure Legend Snippet: Brain-Resident CD4 T Cells Acquire a Residency Phenotype In Situ during a Prolonged Brain Transit (A) Schematic of parabiosis experiments (n = 12,12,18,16,14). (B and C) Curve of best fit for the origin of conventional (B) T cells or (C) Tregs showing CD69 − and CD69 + in the blood and brain. (D) Derived median dwell times. (E and F) t-SNE of conventional (E) T cells and (F) Tregs built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, and CTLA4. FlowSOM clusters represented in color. Host and incoming cells were defined on CD45.1 versus CD45.2 expression, and are shown at the 2-, 4-, and 8-week time points. (G and H) CD69 histograms for CD4 conventional (G) T cells and (H) Tregs. (I and J) Population flow diagrams for conventional (I) T cells and (J) Tregs, in homeostatic state. Circle areas represent population frequencies, calculated independently for blood and brain. Small black circles represent cell death. The size of arrow ends is proportional to the rate of population flow. Numbers display the corresponding entry or exit rate, in events/1,000 cells/day. Numbers with asterisk denote rates with high estimation uncertainty. Population transitions with rates lower than 0.1/1,000 cells/day are not shown. See also Figure S3 .

    Techniques Used: In Situ, Derivative Assay, Expressing

    A Conserved Residency Profile for CD4 T Cells and Regulatory T Cells in the Healthy Mouse and Human Brain, Related to Figure 2 (A) Healthy perfused mouse brains were compared to blood by high-dimensional flow cytometry. Comparison of expression levels of CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4 on blood versus brain CD4 + CD3 + CD45 + CD8 - Foxp3 - T cells or (B) CD4 + CD3 + CD45 + CD8 - Foxp3 + Tregs (n = 5). (C) Transcription profile of CD4 + T cells purified from the murine brain, with analysis through the 10X single cell pipeline and filtering for known cytokines. Naive, activated and regulatory cells were defined based on tSNE clusters and the relative expression of CD44, CD62L and Foxp3 within each cluster. (D) CD4 T cells were assessed in the perfused mouse brain by high-dimensional flow cytometry. Wild-type mice were sampled across the late embryonic (day 19), post-natal development (day 5, 10, 21, 30) and during healthy aging (weeks 8, 12, 30 and 52). n = 9,3,3,5,2,8,5,6,5. Quantification of CD4 + cells per gram of brain tissue, (E) CD4 + T cells, as percentage of CD45 + cells, (F) CD4 + Foxp3 - conventional T cells and (G) CD4 + Foxp3 + regulatory T cells. (H) tSNE of CD4 + Foxp3 - T cells gated on CD4 + Foxp3 - CD3 + CD8 - CD45 + cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4. FlowSOM clusters identified in color. P values refer to cross-entropy difference between age-matched blood and brain samples. Dendrogram represents cross-entropy distance between samples. (I) Comparison of expression levels of CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4 on blood versus brain CD4 + CD3 + CD45 + CD8 - Foxp3 - T cells at different ages. (G) tSNE of CD4 + Foxp3 + T cells gated on CD4 + Foxp3 + CD3 + CD8 - CD45 + cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4. FlowSOM clusters identified in color. P values refer to cross-entropy difference between age-matched blood and brain samples. (J) Comparison of expression levels of CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4 on blood versus brain CD4 + CD3 + CD45 + CD8 - Foxp3 + T cells at different ages. (K) Brain regions were surgically dissected and resident CD4 T cells were characterized by high-dimensional flow cytometry at 10 days, 30 weeks, 60 weeks and 90 weeks of age (n = 6,4,4,5). (L) Numbers and frequencies of CD4 T cells across brain regions in pups and adult mice. (M) tSNE of CD4 + Foxp3 - T cells gated on CD4 + Foxp3 - CD3 + CD8 - CD45 + cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4, shown for blood and different brain regions in adult mice. The adjusted P value reflects the cross-entropy difference between tSNE plots in brain region versus blood. (N) Dendrogram showing the relationship in Tconv across the brain regions based on cross-entropy differences in tSNE. (O) CD69 expression in Tconv from the brain regions in pups and adult mice. (P) Heatmap showing expression of markers in brain region Tconv. (Q) tSNE of CD4 + Foxp3 + T cells gated on CD4 + Foxp3 + CD3 + CD8 - CD45 + cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4, shown for blood and different brain regions. The adjusted P value reflects the cross-entropy difference between tSNE plots in brain region versus blood. (R) CD69 expression in Treg from the brain regions in pups and adult mice. (S) Heatmap showing expression of markers in brain region Treg. (T) Unaffected human brain tissues removed during brain surgery were compared to peripheral blood mononuclear cells by high-dimensional flow cytometry (n = 4). Representative histograms for CD4 + Foxp3 - T cells from human peripheral blood mononuclear cells, white matter, gray matter and meninges for CCR2, CXCR3, PD-1 and CD69. (U) Expression of ICOS, CD28, CD69, Ki-67, CD95, CD31, HLA-DR, CCR2, CXCR5, CD25, PD-1, CXCR3, RORγT, CCR4, CTLA-4, CCR7 and CD45RA on CD4 + Foxp3 - T cells. (V) tSNE of CD4 + Foxp3 + T cells gated on CD4 + Foxp3 + CD3 + CD8 - CD14 - cells and built on ICOS, CD28, CD69, Ki-67, CD95, CD31, HLA-DR, CCR2, CXCR5, CD25, PD-1, CXCR3, RORγT, CCR4, CTLA-4, CCR7 and CD45RA. Colors indicate FlowSOM clusters. Dendrogram showing the relationship across the brain regions based on cross-entropy differences in tSNE. (W) Representative histograms for CD4 + Foxp3 + T cells from human peripheral blood mononuclear cells, white matter, gray matter and meninges for CCR2, CXCR3, PD-1 and CD69. (X) Expression of ICOS, CD28, CD69, Ki-67, CD95, CD31, HLA-DR, CCR2, CXCR5, CD25, PD-1, CXCR3, RORγT, CCR4, CTLA-4, CCR7 and CD45RA on CD4 + Foxp3 + T cells. (Y) Single cell RNaseq data from sorted CD4 + CD3 + CD8 - cells from the human brain and PBMCs. Quality control filtering and gating based on expression markers identified 86 CD4 + T cells from the brain and 567 CD4 + T cells from the blood. tSNE dimensionality reduction is used for cluster display, with lineage marker expression indicated by color for CD3D , CD4 , IL7R , IL2RA , FOXP3 , CD44 , SELL , AREG , CD69 , KLRG1 and NR4A1 . (Z) Differentially expressed genes were assessed for pathway by GSEA.
    Figure Legend Snippet: A Conserved Residency Profile for CD4 T Cells and Regulatory T Cells in the Healthy Mouse and Human Brain, Related to Figure 2 (A) Healthy perfused mouse brains were compared to blood by high-dimensional flow cytometry. Comparison of expression levels of CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4 on blood versus brain CD4 + CD3 + CD45 + CD8 - Foxp3 - T cells or (B) CD4 + CD3 + CD45 + CD8 - Foxp3 + Tregs (n = 5). (C) Transcription profile of CD4 + T cells purified from the murine brain, with analysis through the 10X single cell pipeline and filtering for known cytokines. Naive, activated and regulatory cells were defined based on tSNE clusters and the relative expression of CD44, CD62L and Foxp3 within each cluster. (D) CD4 T cells were assessed in the perfused mouse brain by high-dimensional flow cytometry. Wild-type mice were sampled across the late embryonic (day 19), post-natal development (day 5, 10, 21, 30) and during healthy aging (weeks 8, 12, 30 and 52). n = 9,3,3,5,2,8,5,6,5. Quantification of CD4 + cells per gram of brain tissue, (E) CD4 + T cells, as percentage of CD45 + cells, (F) CD4 + Foxp3 - conventional T cells and (G) CD4 + Foxp3 + regulatory T cells. (H) tSNE of CD4 + Foxp3 - T cells gated on CD4 + Foxp3 - CD3 + CD8 - CD45 + cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4. FlowSOM clusters identified in color. P values refer to cross-entropy difference between age-matched blood and brain samples. Dendrogram represents cross-entropy distance between samples. (I) Comparison of expression levels of CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4 on blood versus brain CD4 + CD3 + CD45 + CD8 - Foxp3 - T cells at different ages. (G) tSNE of CD4 + Foxp3 + T cells gated on CD4 + Foxp3 + CD3 + CD8 - CD45 + cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4. FlowSOM clusters identified in color. P values refer to cross-entropy difference between age-matched blood and brain samples. (J) Comparison of expression levels of CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4 on blood versus brain CD4 + CD3 + CD45 + CD8 - Foxp3 + T cells at different ages. (K) Brain regions were surgically dissected and resident CD4 T cells were characterized by high-dimensional flow cytometry at 10 days, 30 weeks, 60 weeks and 90 weeks of age (n = 6,4,4,5). (L) Numbers and frequencies of CD4 T cells across brain regions in pups and adult mice. (M) tSNE of CD4 + Foxp3 - T cells gated on CD4 + Foxp3 - CD3 + CD8 - CD45 + cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4, shown for blood and different brain regions in adult mice. The adjusted P value reflects the cross-entropy difference between tSNE plots in brain region versus blood. (N) Dendrogram showing the relationship in Tconv across the brain regions based on cross-entropy differences in tSNE. (O) CD69 expression in Tconv from the brain regions in pups and adult mice. (P) Heatmap showing expression of markers in brain region Tconv. (Q) tSNE of CD4 + Foxp3 + T cells gated on CD4 + Foxp3 + CD3 + CD8 - CD45 + cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4, shown for blood and different brain regions. The adjusted P value reflects the cross-entropy difference between tSNE plots in brain region versus blood. (R) CD69 expression in Treg from the brain regions in pups and adult mice. (S) Heatmap showing expression of markers in brain region Treg. (T) Unaffected human brain tissues removed during brain surgery were compared to peripheral blood mononuclear cells by high-dimensional flow cytometry (n = 4). Representative histograms for CD4 + Foxp3 - T cells from human peripheral blood mononuclear cells, white matter, gray matter and meninges for CCR2, CXCR3, PD-1 and CD69. (U) Expression of ICOS, CD28, CD69, Ki-67, CD95, CD31, HLA-DR, CCR2, CXCR5, CD25, PD-1, CXCR3, RORγT, CCR4, CTLA-4, CCR7 and CD45RA on CD4 + Foxp3 - T cells. (V) tSNE of CD4 + Foxp3 + T cells gated on CD4 + Foxp3 + CD3 + CD8 - CD14 - cells and built on ICOS, CD28, CD69, Ki-67, CD95, CD31, HLA-DR, CCR2, CXCR5, CD25, PD-1, CXCR3, RORγT, CCR4, CTLA-4, CCR7 and CD45RA. Colors indicate FlowSOM clusters. Dendrogram showing the relationship across the brain regions based on cross-entropy differences in tSNE. (W) Representative histograms for CD4 + Foxp3 + T cells from human peripheral blood mononuclear cells, white matter, gray matter and meninges for CCR2, CXCR3, PD-1 and CD69. (X) Expression of ICOS, CD28, CD69, Ki-67, CD95, CD31, HLA-DR, CCR2, CXCR5, CD25, PD-1, CXCR3, RORγT, CCR4, CTLA-4, CCR7 and CD45RA on CD4 + Foxp3 + T cells. (Y) Single cell RNaseq data from sorted CD4 + CD3 + CD8 - cells from the human brain and PBMCs. Quality control filtering and gating based on expression markers identified 86 CD4 + T cells from the brain and 567 CD4 + T cells from the blood. tSNE dimensionality reduction is used for cluster display, with lineage marker expression indicated by color for CD3D , CD4 , IL7R , IL2RA , FOXP3 , CD44 , SELL , AREG , CD69 , KLRG1 and NR4A1 . (Z) Differentially expressed genes were assessed for pathway by GSEA.

    Techniques Used: Flow Cytometry, Expressing, Purification, Mouse Assay, Marker

    CD4 T Cells Are Present in the Healthy Mouse and Human Brain For a Figure360 author presentation of this figure, see https://doi.org/10.1016/j.cell.2020.06.026 . (A and B) Surface rendering of confocal images. Representative image of (A) CD4 T cell crossing laminin 4 barrier or (B) laminin α1 barrier within midbrain meningeal folds. (C) Representative image of a CD4 T cell undergoing transvascular movement in the hindbrain. (D–F) Representative images of CD4 T cells beyond the laminin 4/α1 barrier in the (D) cerebellum, (E) hindbrain, or (F) olfactory bulb. (G–I) Representative images of CD4 T cells (G) enclosed by the glia limitans in the mid-brain and beyond the glia limitans in (H) the midbrain and (I) the cerebellum. (J and K) Representative images of CD4 T cells in close proximity to microglia in the (J) midbrain (K) or hindbrain. Scale bar, 20 μm. (L) Quantification of CD4 T cells based on proximity to vasculature from the sagittal sections of the mouse brain. (M) Relative and absolute distribution of CD4 T cells across mouse brain regions, based on quantification of sagittal sections. Values represent the number of non-vascular CD4 T cells located in each region, per mm 3 or in absolute number (biological replicates from the average of 21–23 quantified sections). (N) Mice were intravenously (i.v.) injected with anti-CD45-PE and perfused. Brains were then dissected and analyzed by flow cytometry for the proportion of intravascular CD4 T cells (n = 3,5,2). (O and P) CD4 T cells in dissected mouse brain regions by flow cytometry, based on (O) percentage of CD45 + cells or (P) absolute number (n = 4). (Q and R) Absolute number of (Q) CD4 T cells and (R) Tregs in the perfused healthy mouse brain, as assessed by imaging (excluding vascular cells) and flow cytometry (excluding meningeal cells). (S) Average proportion of CD4 T cells in the white matter, gray matter, and meninges of healthy brain tissue (n = 4). See also Figure S1 and Videos S1 , S2 , S3 , S4 , S5 , S6 , and S7 .
    Figure Legend Snippet: CD4 T Cells Are Present in the Healthy Mouse and Human Brain For a Figure360 author presentation of this figure, see https://doi.org/10.1016/j.cell.2020.06.026 . (A and B) Surface rendering of confocal images. Representative image of (A) CD4 T cell crossing laminin 4 barrier or (B) laminin α1 barrier within midbrain meningeal folds. (C) Representative image of a CD4 T cell undergoing transvascular movement in the hindbrain. (D–F) Representative images of CD4 T cells beyond the laminin 4/α1 barrier in the (D) cerebellum, (E) hindbrain, or (F) olfactory bulb. (G–I) Representative images of CD4 T cells (G) enclosed by the glia limitans in the mid-brain and beyond the glia limitans in (H) the midbrain and (I) the cerebellum. (J and K) Representative images of CD4 T cells in close proximity to microglia in the (J) midbrain (K) or hindbrain. Scale bar, 20 μm. (L) Quantification of CD4 T cells based on proximity to vasculature from the sagittal sections of the mouse brain. (M) Relative and absolute distribution of CD4 T cells across mouse brain regions, based on quantification of sagittal sections. Values represent the number of non-vascular CD4 T cells located in each region, per mm 3 or in absolute number (biological replicates from the average of 21–23 quantified sections). (N) Mice were intravenously (i.v.) injected with anti-CD45-PE and perfused. Brains were then dissected and analyzed by flow cytometry for the proportion of intravascular CD4 T cells (n = 3,5,2). (O and P) CD4 T cells in dissected mouse brain regions by flow cytometry, based on (O) percentage of CD45 + cells or (P) absolute number (n = 4). (Q and R) Absolute number of (Q) CD4 T cells and (R) Tregs in the perfused healthy mouse brain, as assessed by imaging (excluding vascular cells) and flow cytometry (excluding meningeal cells). (S) Average proportion of CD4 T cells in the white matter, gray matter, and meninges of healthy brain tissue (n = 4). See also Figure S1 and Videos S1 , S2 , S3 , S4 , S5 , S6 , and S7 .

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

    Parabiotic Analysis of Brain T Cell Kinetics, Related to Figure 3 Parabiosis surgery was performed on Foxp3 Thy1.1 CD45.1 and Foxp3 Thy1.1 CD45.2 mice, with analysis of brain and blood CD4 T cells at 1, 2, 4, 8 and 12 weeks (n = 12, 12, 18, 16, 14). Curves of best fit as well as the mean ± SEM for each subset are displayed at each time-point. (A) Curve of best fit for the origin of CD4 + Foxp3 - conventional T cells in the blood and brain. (B) Curve of best fit for the origin of CD4 + Foxp3 - conventional T cells in the blood and brain, divided into naive (CD62L hi CD44 low ) and antigen-experienced (CD62L low CD44 hi ) subsets. (C) Curve of best fit for the origin of CD4 + Foxp3 + regulatory T cells in the blood and brain, divided into CD69 - and CD69 + subsets in the brain. (D) Curve of best fit for the origin of CD4 + Foxp3 + regulatory T cells in the blood and brain, divided into naive (CD62L hi CD44 low ) and antigen-experienced (CD62L low CD44 hi ) subsets. (E) Curve of best fit for the origin of CD4 + Foxp3 + regulatory T cells in the blood and brain, divided into thymic-derived (Nrp1 + ) and peripherally-derived (Nrp1 - ) subsets. (F) Time evolution of average populations as predicted by a continuous-time Markov chain model fitted on the data from weeks 2 to 12, with homeostatic state identified from week 0. Experimental data (points) and calculations (solid lines) are shown in blue for host cells and in red for donor cells. Big points identify experimental population averages, whereas homeostatic states are displayed with black dashed lines. Model for CD4 + Foxp3 - conventional T cells and (G) CD4 + Foxp3 + regulatory T cells. Tissue and subpopulation for each graph are shown in the top-left graph label.
    Figure Legend Snippet: Parabiotic Analysis of Brain T Cell Kinetics, Related to Figure 3 Parabiosis surgery was performed on Foxp3 Thy1.1 CD45.1 and Foxp3 Thy1.1 CD45.2 mice, with analysis of brain and blood CD4 T cells at 1, 2, 4, 8 and 12 weeks (n = 12, 12, 18, 16, 14). Curves of best fit as well as the mean ± SEM for each subset are displayed at each time-point. (A) Curve of best fit for the origin of CD4 + Foxp3 - conventional T cells in the blood and brain. (B) Curve of best fit for the origin of CD4 + Foxp3 - conventional T cells in the blood and brain, divided into naive (CD62L hi CD44 low ) and antigen-experienced (CD62L low CD44 hi ) subsets. (C) Curve of best fit for the origin of CD4 + Foxp3 + regulatory T cells in the blood and brain, divided into CD69 - and CD69 + subsets in the brain. (D) Curve of best fit for the origin of CD4 + Foxp3 + regulatory T cells in the blood and brain, divided into naive (CD62L hi CD44 low ) and antigen-experienced (CD62L low CD44 hi ) subsets. (E) Curve of best fit for the origin of CD4 + Foxp3 + regulatory T cells in the blood and brain, divided into thymic-derived (Nrp1 + ) and peripherally-derived (Nrp1 - ) subsets. (F) Time evolution of average populations as predicted by a continuous-time Markov chain model fitted on the data from weeks 2 to 12, with homeostatic state identified from week 0. Experimental data (points) and calculations (solid lines) are shown in blue for host cells and in red for donor cells. Big points identify experimental population averages, whereas homeostatic states are displayed with black dashed lines. Model for CD4 + Foxp3 - conventional T cells and (G) CD4 + Foxp3 + regulatory T cells. Tissue and subpopulation for each graph are shown in the top-left graph label.

    Techniques Used: Mouse Assay, Derivative Assay

    9) Product Images from "γδT Cells Are Prevalent in the Proximal Aorta and Drive Nascent Atherosclerotic Lesion Progression and Neutrophilia in Hypercholesterolemic Mice"

    Article Title: γδT Cells Are Prevalent in the Proximal Aorta and Drive Nascent Atherosclerotic Lesion Progression and Neutrophilia in Hypercholesterolemic Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0109416

    Increased γδT cells in proximal aorta of ApoE KO vs. B6 mice. Single-cell suspensions from enzyme-digested aortas of 22 wk-old chow-fed mice were stained with anti-CD3-FITC, anti-CD45-PE and anti-TCRγδ-APC and analyzed by FACS. A: Gating strategy and representative FACS plots. B: Bar s indicate means; symbols indicate the absolute number of aortic T cells in individual mice. γδT cells (CD3 + TCRγδ + ) were significantly increased in ApoE KO vs. B6 aorta (* p
    Figure Legend Snippet: Increased γδT cells in proximal aorta of ApoE KO vs. B6 mice. Single-cell suspensions from enzyme-digested aortas of 22 wk-old chow-fed mice were stained with anti-CD3-FITC, anti-CD45-PE and anti-TCRγδ-APC and analyzed by FACS. A: Gating strategy and representative FACS plots. B: Bar s indicate means; symbols indicate the absolute number of aortic T cells in individual mice. γδT cells (CD3 + TCRγδ + ) were significantly increased in ApoE KO vs. B6 aorta (* p

    Techniques Used: Mouse Assay, Staining, FACS

    10) Product Images from "Fetal hematopoietic stem cell homing is controlled by VEGF regulating the integrity and oxidative status of the stromal-vascular bone marrow niches"

    Article Title: Fetal hematopoietic stem cell homing is controlled by VEGF regulating the integrity and oxidative status of the stromal-vascular bone marrow niches

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2021.109618

    Altered dynamics and destination of fetal HSPC trafficking in VEGF cTg mice (A and B) Flow cytometry of LSK, LSK-SLAM, and CD45 + cell populations (n = 5–7). (C and D) Frequency of LSK, LSK-SLAM, and CD45 + cells as % of all counted cells (n = 5–6). (E) Frequency of circulating LSK cells (relative to all CD45 + cells) (n = 5–9). (F and G) LSK, LSK-SLAM, and CD45 + cell counts in (F) spleen and (G) liver (n = 5–7). (H) Flow cytometry of megakaryocyte progenitors (MkP), pre-colony forming unit – erythroid (preCFU-E), and pre-granulocytes and monocytes (preGM cells) in liver (n = 3). (I) Angiopoietin-like 3 ( ANGPTL3 ) and thrombopoietin ( TPO ) qRT-PCR in E18.5 livers (n = 8). Graphs, mean ± SEM, and individual data points; ∗ p
    Figure Legend Snippet: Altered dynamics and destination of fetal HSPC trafficking in VEGF cTg mice (A and B) Flow cytometry of LSK, LSK-SLAM, and CD45 + cell populations (n = 5–7). (C and D) Frequency of LSK, LSK-SLAM, and CD45 + cells as % of all counted cells (n = 5–6). (E) Frequency of circulating LSK cells (relative to all CD45 + cells) (n = 5–9). (F and G) LSK, LSK-SLAM, and CD45 + cell counts in (F) spleen and (G) liver (n = 5–7). (H) Flow cytometry of megakaryocyte progenitors (MkP), pre-colony forming unit – erythroid (preCFU-E), and pre-granulocytes and monocytes (preGM cells) in liver (n = 3). (I) Angiopoietin-like 3 ( ANGPTL3 ) and thrombopoietin ( TPO ) qRT-PCR in E18.5 livers (n = 8). Graphs, mean ± SEM, and individual data points; ∗ p

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

    Delayed-onset VEGF overexpression in developing bones creates a hypomorph model of osteo-angiogenic alterations with a normal-size BM cavity (A) Total VEGF mRNA and VEGF 164 protein levels at E18.5 and P2 (n = 6–11). (B) VEGF serum levels at E18.5 (n = 6). (C) E18.5 skeletal preparations; bottom, hindlimbs. (D) H E and BM cavity length of E18.5 tibias (n = 3). (E–G) Micro-CT of E18.5 tibias with (F) bone volume/tissue volume (BV/TV) and (G) BM cavity volume (n = 5). (H and I) CD31 IHC and (I) histomorphometry on E18.5 tibias. BLV, blood vessel (n = 3–4). (J) MIP (15-μm depth) of Osx--Cre:GFP, Emcn, and Hoechst signals in P2 tibias. (K) Flow cytometry of CD45 − Ter119 − CD31 − Osx-Cre:GFP + cells in P2 limbs (n = 7). (L and M) LSK, LSK-SLAM, and CD45 + cell counts in (L) bones and (M) spleens (n = 3–8). Graphs, mean ± SEM; ∗ p
    Figure Legend Snippet: Delayed-onset VEGF overexpression in developing bones creates a hypomorph model of osteo-angiogenic alterations with a normal-size BM cavity (A) Total VEGF mRNA and VEGF 164 protein levels at E18.5 and P2 (n = 6–11). (B) VEGF serum levels at E18.5 (n = 6). (C) E18.5 skeletal preparations; bottom, hindlimbs. (D) H E and BM cavity length of E18.5 tibias (n = 3). (E–G) Micro-CT of E18.5 tibias with (F) bone volume/tissue volume (BV/TV) and (G) BM cavity volume (n = 5). (H and I) CD31 IHC and (I) histomorphometry on E18.5 tibias. BLV, blood vessel (n = 3–4). (J) MIP (15-μm depth) of Osx--Cre:GFP, Emcn, and Hoechst signals in P2 tibias. (K) Flow cytometry of CD45 − Ter119 − CD31 − Osx-Cre:GFP + cells in P2 limbs (n = 7). (L and M) LSK, LSK-SLAM, and CD45 + cell counts in (L) bones and (M) spleens (n = 3–8). Graphs, mean ± SEM; ∗ p

    Techniques Used: Over Expression, Micro-CT, Immunohistochemistry, Flow Cytometry

    Osteo-angiogenic development of long bones is disrupted in VEGF cTg mice (A) Total VEGF mRNA (qRT-PCR) and VEGF 164 protein levels (ELISA) in bones of control and VEGF cTg embryos (n = 4–8). (B) VEGF serum levels (ELISA) (n = 6). (C) Skeletal preparations at E18.5. Middle panels, dissected hindlimbs of control (top) and VEGF cTg (bottom). (D) Longitudinal (top) and transversal (bottom) 3D micro-computed tomography (micro-CT) reconstructions of E18.5 tibias. (E) Histology of E18.5 tibias; (left) Safranin O and (right) H E. Brackets, BM cavity. (F) BM cavity volume at E18.5 (n = 7). (G) CD31 IHC on tibia sections. Arrows, aberrant perichondrial vascularization. (H) Maximum intensity projection (MIP) of 20-μm depth confocal imaging of Osx-Cre:GFP (nuclear green, OPCs), Emcn (red, blood vessels), and Hoechst (blue, nuclei) in E18.5 tibias. Left, overview; middle, metaphysis; right, diaphysis. (I) Flow cytometry of CD45 − Ter119 − CD31 − Osx/GFP + cells in E18.5 bones (n = 4–5). All graphs represent mean ± SEM and depict individual data points derived from different mice (also in the next figures); ∗ p
    Figure Legend Snippet: Osteo-angiogenic development of long bones is disrupted in VEGF cTg mice (A) Total VEGF mRNA (qRT-PCR) and VEGF 164 protein levels (ELISA) in bones of control and VEGF cTg embryos (n = 4–8). (B) VEGF serum levels (ELISA) (n = 6). (C) Skeletal preparations at E18.5. Middle panels, dissected hindlimbs of control (top) and VEGF cTg (bottom). (D) Longitudinal (top) and transversal (bottom) 3D micro-computed tomography (micro-CT) reconstructions of E18.5 tibias. (E) Histology of E18.5 tibias; (left) Safranin O and (right) H E. Brackets, BM cavity. (F) BM cavity volume at E18.5 (n = 7). (G) CD31 IHC on tibia sections. Arrows, aberrant perichondrial vascularization. (H) Maximum intensity projection (MIP) of 20-μm depth confocal imaging of Osx-Cre:GFP (nuclear green, OPCs), Emcn (red, blood vessels), and Hoechst (blue, nuclei) in E18.5 tibias. Left, overview; middle, metaphysis; right, diaphysis. (I) Flow cytometry of CD45 − Ter119 − CD31 − Osx/GFP + cells in E18.5 bones (n = 4–5). All graphs represent mean ± SEM and depict individual data points derived from different mice (also in the next figures); ∗ p

    Techniques Used: Mouse Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Micro-CT, Immunohistochemistry, Imaging, Flow Cytometry, Derivative Assay

    Responses of the stromal-vascular BM compartment to increased VEGF (A) Flow cytometry analysis of ROS levels by conversion of the probe dichlorodihydrofluorescein diacetate (DCFDA) to the highly fluorescent 2′,7′-di-chlorofluorescein (DCF), in (left) ECs (CD45 − Ter119 − CD31 + ) and (right) hematopoietic cells (CD45 + and/or Ter119 + ) from E16.5 bones (n = 5–7). (B) DCF levels representing ROS in ECs of (left) arteriolar (CD45 − Ter119 − CD31 + Sca-1 + ) and (right) sinusoidal (CD45 − Ter119 − CD31 + Sca-1 − ) subtypes, derived from P2 bones (n = 5–7). (C) MIP (100-μm z axis) of CD31 and Emcn signals in E18.5 tibia BM cavities (dotted lines). Left, overviews; right, metaphyseal (top) and diaphyseal (bottom) regions. (D) Directionality of blood vessels quantified from segmented and skeletonized CD31 staining. Angle 0° = vessel running along the longitudinal bone axis; 90° = parallel to the growth plate zones. Right, area under the curve (AUC) for vessels with directionality between −45° and 45° (n = 4). (E and F) Number of blood vessels per mm 2 (E) and distribution of vessels according to width (F), quantified on segmented CD31 staining images (n = 4). (G) MIP (60-μm depth) of CD31 and Emcn stained tibias at P2. (H) EC counts by flow cytometry (n = 4–8). (I) IHC for phospho-histone H3 (PHH3) and Emcn at P2, and Emcn + PHH3 + mitotic ECs quantified in the dashed areas of the metaphysis and diaphysis (n = 3). (J) Proportions sinusoidal (CD31 + Sca-1 − ) and arteriolar (CD31 + Sca-1 + ) ECs (n = 4–8). (K) MIP spanning 100-μm tissue depth of CD31, Emcn, and Osx-Cre:GFP signals on E18.5 tibias. Also see Video S1 . (L) Nearest-neighbor distance analysis showing for distinct Osx-Cre:GFP + cells the distance to the closest CD31 + or Emcn + signal. Plots display individual values, median, and interquartile range (no statistics applied). (M) Proportion of triple-negative (TN; CD45 − Ter119 − CD31 − ) CD51 + Sca-1 − and CD51 + Sca-1 + osteolineage progenitors in E18.5 limbs (n = 4–5). (N) TN PαS (PDGFRα + Sca-1 + ) and PDGFRβ + cell numbers in (left) E18.5 and (right) P2 bones (n = 4–7). (O) MIP (20-μm depth) of PDGFRβ, Emcn, and Hoechst signals in (top) E18.5 and (bottom) P2 tibias. (P) TEM of E16.5 tibias. Dotted lines, blood vessels; asterisks, lumens; orange arrows, irregular ruffled membranes; blue arrowheads, pinocytotic vesicles; green block arrows, mitochondria; red arrowheads, fenestrations/pores between zones of tight junctions (#). Graphs, mean ± SEM, except in (L). ∗ p
    Figure Legend Snippet: Responses of the stromal-vascular BM compartment to increased VEGF (A) Flow cytometry analysis of ROS levels by conversion of the probe dichlorodihydrofluorescein diacetate (DCFDA) to the highly fluorescent 2′,7′-di-chlorofluorescein (DCF), in (left) ECs (CD45 − Ter119 − CD31 + ) and (right) hematopoietic cells (CD45 + and/or Ter119 + ) from E16.5 bones (n = 5–7). (B) DCF levels representing ROS in ECs of (left) arteriolar (CD45 − Ter119 − CD31 + Sca-1 + ) and (right) sinusoidal (CD45 − Ter119 − CD31 + Sca-1 − ) subtypes, derived from P2 bones (n = 5–7). (C) MIP (100-μm z axis) of CD31 and Emcn signals in E18.5 tibia BM cavities (dotted lines). Left, overviews; right, metaphyseal (top) and diaphyseal (bottom) regions. (D) Directionality of blood vessels quantified from segmented and skeletonized CD31 staining. Angle 0° = vessel running along the longitudinal bone axis; 90° = parallel to the growth plate zones. Right, area under the curve (AUC) for vessels with directionality between −45° and 45° (n = 4). (E and F) Number of blood vessels per mm 2 (E) and distribution of vessels according to width (F), quantified on segmented CD31 staining images (n = 4). (G) MIP (60-μm depth) of CD31 and Emcn stained tibias at P2. (H) EC counts by flow cytometry (n = 4–8). (I) IHC for phospho-histone H3 (PHH3) and Emcn at P2, and Emcn + PHH3 + mitotic ECs quantified in the dashed areas of the metaphysis and diaphysis (n = 3). (J) Proportions sinusoidal (CD31 + Sca-1 − ) and arteriolar (CD31 + Sca-1 + ) ECs (n = 4–8). (K) MIP spanning 100-μm tissue depth of CD31, Emcn, and Osx-Cre:GFP signals on E18.5 tibias. Also see Video S1 . (L) Nearest-neighbor distance analysis showing for distinct Osx-Cre:GFP + cells the distance to the closest CD31 + or Emcn + signal. Plots display individual values, median, and interquartile range (no statistics applied). (M) Proportion of triple-negative (TN; CD45 − Ter119 − CD31 − ) CD51 + Sca-1 − and CD51 + Sca-1 + osteolineage progenitors in E18.5 limbs (n = 4–5). (N) TN PαS (PDGFRα + Sca-1 + ) and PDGFRβ + cell numbers in (left) E18.5 and (right) P2 bones (n = 4–7). (O) MIP (20-μm depth) of PDGFRβ, Emcn, and Hoechst signals in (top) E18.5 and (bottom) P2 tibias. (P) TEM of E16.5 tibias. Dotted lines, blood vessels; asterisks, lumens; orange arrows, irregular ruffled membranes; blue arrowheads, pinocytotic vesicles; green block arrows, mitochondria; red arrowheads, fenestrations/pores between zones of tight junctions (#). Graphs, mean ± SEM, except in (L). ∗ p

    Techniques Used: Flow Cytometry, Derivative Assay, Staining, Immunohistochemistry, Transmission Electron Microscopy, Blocking Assay

    11) Product Images from "Platelets supply p38 MAPK signaling that licenses pro-inflammatory cytokine responses of human monocytes"

    Article Title: Platelets supply p38 MAPK signaling that licenses pro-inflammatory cytokine responses of human monocytes

    Journal: bioRxiv

    doi: 10.1101/2022.08.10.503291

    The platelet-monocyte crosstalk is contact dependent. ( A ) Immunoblot of MAPK14 (p38) or phosphorylated MAPK14 (P-p38) in resting (Unstim) or LPS-activated (LPS) human platelets. Platelets were submitted to centrifugation at 500 x g, 3000 x g or 10,000 x g and the levels of MAPK14 were assessed in the pellets (WCL) or supernatants (CSF) after centrifugation. Results are representative of two independent experiments. ( B ) Cytokine levels in CFS of StdMo, PdMo, and PdMo that were supplemented with CSF of platelets generated as in A . ( C ) IL-1β levels in CFS of StdMo, PdMo, and PdMo that were supplemented with autologous platelets (PdMo + Plts) directly, or in trans-well separated by a 0.4 μm membrane. IL-1β were measured by HTRF and are represented as floating bar graphs. Additional cytokines are displayed in Figure S6 . ( D ) Confocal imaging of StdMo and platelets. Cells were stained with CD41a AF488 (platelets: green) and CD14 AF647 (monocytes: red). Nuclei were stained with Hoechst 34580 (blue). Scale: 24 μm (top panel); 4.8 μm (bottom panel). White arrows indicate points of contacts between platelets and monocytes. Images are from one representative of four independent experiments. ( E ) Representative flow cytometry assessment and gating strategy of StdMo that were incubated with growing concentrations of KPL-1 (5, 10 or 25 μg ml -1 ), or IgG (25 μg ml -1 ). Gates indicate the frequencies of free platelets (CD45 - CD41a + ), MPAs (CD45 + CD41a + ) and platelet-free monocytes (CD45 + CD41a - ). Data is representative of two independent experiments with several donors. ( F ) Cumulative flow cytometry frequency of free monocytes (CD45 + CD41a - ) in StdMo treated with growing concentrations of KPL-1 (5, 10 or 25 μg ml -1 ), a specific mAb against P-selectin, or an unrelated isotype IgG control (25 μg ml -1 ). See Figure S4E for gating strategy. ( G ) IL-1β, TNFα and IL-6 levels released by stimulated StdMo that were treated with increasing concentrations of KPL-1 (5, 10 or 25 μg ml -1 ), or IgG (25 μg ml -1 ). Data is displayed as floating bars with the max/min values and mean (white bands). P values were calculated with 2-Way Anova, Tukey’s multiple comparison test, and are displayed in the figure. Each symbol represents one independent experiment or blood donor. ( H ) IL-1β and TNFα levels in CFS of StdMo, PdMo, and PdMo that were supplemented with autologous platelets (PdMo + Plts, 100:1 platelets/ monocytes). Monocytes were pre-treated with Cytochalasin D (CytoD, 50 μM) or Dynasore (DS, 30 μM) before being supplied with Plts. Cells were stimulated with LPS (2 ng·ml -1 ), followed by activation with nigericin (10 μM).
    Figure Legend Snippet: The platelet-monocyte crosstalk is contact dependent. ( A ) Immunoblot of MAPK14 (p38) or phosphorylated MAPK14 (P-p38) in resting (Unstim) or LPS-activated (LPS) human platelets. Platelets were submitted to centrifugation at 500 x g, 3000 x g or 10,000 x g and the levels of MAPK14 were assessed in the pellets (WCL) or supernatants (CSF) after centrifugation. Results are representative of two independent experiments. ( B ) Cytokine levels in CFS of StdMo, PdMo, and PdMo that were supplemented with CSF of platelets generated as in A . ( C ) IL-1β levels in CFS of StdMo, PdMo, and PdMo that were supplemented with autologous platelets (PdMo + Plts) directly, or in trans-well separated by a 0.4 μm membrane. IL-1β were measured by HTRF and are represented as floating bar graphs. Additional cytokines are displayed in Figure S6 . ( D ) Confocal imaging of StdMo and platelets. Cells were stained with CD41a AF488 (platelets: green) and CD14 AF647 (monocytes: red). Nuclei were stained with Hoechst 34580 (blue). Scale: 24 μm (top panel); 4.8 μm (bottom panel). White arrows indicate points of contacts between platelets and monocytes. Images are from one representative of four independent experiments. ( E ) Representative flow cytometry assessment and gating strategy of StdMo that were incubated with growing concentrations of KPL-1 (5, 10 or 25 μg ml -1 ), or IgG (25 μg ml -1 ). Gates indicate the frequencies of free platelets (CD45 - CD41a + ), MPAs (CD45 + CD41a + ) and platelet-free monocytes (CD45 + CD41a - ). Data is representative of two independent experiments with several donors. ( F ) Cumulative flow cytometry frequency of free monocytes (CD45 + CD41a - ) in StdMo treated with growing concentrations of KPL-1 (5, 10 or 25 μg ml -1 ), a specific mAb against P-selectin, or an unrelated isotype IgG control (25 μg ml -1 ). See Figure S4E for gating strategy. ( G ) IL-1β, TNFα and IL-6 levels released by stimulated StdMo that were treated with increasing concentrations of KPL-1 (5, 10 or 25 μg ml -1 ), or IgG (25 μg ml -1 ). Data is displayed as floating bars with the max/min values and mean (white bands). P values were calculated with 2-Way Anova, Tukey’s multiple comparison test, and are displayed in the figure. Each symbol represents one independent experiment or blood donor. ( H ) IL-1β and TNFα levels in CFS of StdMo, PdMo, and PdMo that were supplemented with autologous platelets (PdMo + Plts, 100:1 platelets/ monocytes). Monocytes were pre-treated with Cytochalasin D (CytoD, 50 μM) or Dynasore (DS, 30 μM) before being supplied with Plts. Cells were stimulated with LPS (2 ng·ml -1 ), followed by activation with nigericin (10 μM).

    Techniques Used: Centrifugation, Generated, Imaging, Staining, Flow Cytometry, Incubation, Activation Assay

    12) Product Images from "Time-resolved single-cell transcriptomics uncovers dynamics of cardiac neutrophil diversity in murine myocardial infarction"

    Article Title: Time-resolved single-cell transcriptomics uncovers dynamics of cardiac neutrophil diversity in murine myocardial infarction

    Journal: bioRxiv

    doi: 10.1101/738005

    Time-dependent cardiac neutrophil subsets differentially express maturation and activation markers. A ) Log normalized expression levels of the indicated transcripts projected onto the tSNE plot; B) top: gating of LY6G + LY6C int neutrophils and LY6G − LY6C hi monocytes in viable CD45+CD11B+ cells isolated from mechanically dissociated cardiac tissue; bottom: representative dot plots showing ICAM1 expression in LY6C hi monocytes (orange) and neutrophils (purple) at Day 1 and Day 3 post-MI; C) representative histogram of ICAM1 expression and quantification of ICAM1+ cells and fluorescence intensity (Geometric Mean) in the indicated neutrophil subsets; D) representative histograms and quantification of fluorescence intensity (Geometric Mean) for the indicated epitopes on cardiac neutrophil subsets at day 1 (total neutrophils) and day 3 (SIGLECF hi and SIGLECF lo neutrophils). *p
    Figure Legend Snippet: Time-dependent cardiac neutrophil subsets differentially express maturation and activation markers. A ) Log normalized expression levels of the indicated transcripts projected onto the tSNE plot; B) top: gating of LY6G + LY6C int neutrophils and LY6G − LY6C hi monocytes in viable CD45+CD11B+ cells isolated from mechanically dissociated cardiac tissue; bottom: representative dot plots showing ICAM1 expression in LY6C hi monocytes (orange) and neutrophils (purple) at Day 1 and Day 3 post-MI; C) representative histogram of ICAM1 expression and quantification of ICAM1+ cells and fluorescence intensity (Geometric Mean) in the indicated neutrophil subsets; D) representative histograms and quantification of fluorescence intensity (Geometric Mean) for the indicated epitopes on cardiac neutrophil subsets at day 1 (total neutrophils) and day 3 (SIGLECF hi and SIGLECF lo neutrophils). *p

    Techniques Used: Activation Assay, Expressing, Isolation, Fluorescence

    Analysis of time series scRNA-seq of total cardiac CD45+ cells, day 0 to 7 post-MI. A) Summary of the experimental design; B) identification of major myeloid cell lineages in the dataset projected onte the tSNE plot; C) Percent of transcripts encoding ribosomal proteins projected onto the tSNE plot of total myeloid cells; D) violin plot showing percent of transcripts encoding ribosomal proteins in neutrophils, according to time point of origin; E) time point of origin of single neutrophils projected onto the tSNE plot; F) identification of neutrophil clusters on the tSNE plot; G) violin plot showing log normalized expression of the indicated transcripts in neutrophils, according to cluster.
    Figure Legend Snippet: Analysis of time series scRNA-seq of total cardiac CD45+ cells, day 0 to 7 post-MI. A) Summary of the experimental design; B) identification of major myeloid cell lineages in the dataset projected onte the tSNE plot; C) Percent of transcripts encoding ribosomal proteins projected onto the tSNE plot of total myeloid cells; D) violin plot showing percent of transcripts encoding ribosomal proteins in neutrophils, according to time point of origin; E) time point of origin of single neutrophils projected onto the tSNE plot; F) identification of neutrophil clusters on the tSNE plot; G) violin plot showing log normalized expression of the indicated transcripts in neutrophils, according to cluster.

    Techniques Used: Expressing

    Additional flow cytometry analyses. A) Fluorescence minus one (FMO) validation of SIGLECF expression by cardiac neutrophils at Day 3 after MI; B) number of total neutrophils and SIGLECF hi neutrophils in cardiac tissue from non-operated controls (Ctrl), sham and MI operated mice at Day 1 and 3 after myocardial infarction. To analyze remote, non ischemic myocardium, cardiac tissue was sampled above the ligation; C) detection of LY6G neg SIGLECF hi eosinophils, LY6G + SIGLECF hi neutrophils and LY6G + SIGLECF low neutrophils in mechanically digested cardiac tissue at 1 and 3 days post MI (complementary to enzymatic digestion data in Figure 2 ).
    Figure Legend Snippet: Additional flow cytometry analyses. A) Fluorescence minus one (FMO) validation of SIGLECF expression by cardiac neutrophils at Day 3 after MI; B) number of total neutrophils and SIGLECF hi neutrophils in cardiac tissue from non-operated controls (Ctrl), sham and MI operated mice at Day 1 and 3 after myocardial infarction. To analyze remote, non ischemic myocardium, cardiac tissue was sampled above the ligation; C) detection of LY6G neg SIGLECF hi eosinophils, LY6G + SIGLECF hi neutrophils and LY6G + SIGLECF low neutrophils in mechanically digested cardiac tissue at 1 and 3 days post MI (complementary to enzymatic digestion data in Figure 2 ).

    Techniques Used: Flow Cytometry, Fluorescence, Expressing, Mouse Assay, Ligation

    Reanalysis of King et al., scRNA-seq of CD45+ cells at day 4 after MI (King et al. Nat Med 2017). A) tSNE representation of scRNA-seq gene expression data and clustering analysis; B) heatmap of the top 5 marker genes (ordered by Log2 fold change) in each cluster; C) expression of the indicated markers identifies cells corresponding to neutrophils ( S100a8+ , Cxcr2+ , no expression of Adgre1 or Csf1r ) and D) expression of the indicated genes in the two neutrophil clusters (Cluster 3 and 6 on the tSNE plot/heatmap, panel A ).
    Figure Legend Snippet: Reanalysis of King et al., scRNA-seq of CD45+ cells at day 4 after MI (King et al. Nat Med 2017). A) tSNE representation of scRNA-seq gene expression data and clustering analysis; B) heatmap of the top 5 marker genes (ordered by Log2 fold change) in each cluster; C) expression of the indicated markers identifies cells corresponding to neutrophils ( S100a8+ , Cxcr2+ , no expression of Adgre1 or Csf1r ) and D) expression of the indicated genes in the two neutrophil clusters (Cluster 3 and 6 on the tSNE plot/heatmap, panel A ).

    Techniques Used: Expressing, Marker

    Circulating, bone marrow and splenic neutrophils do not acquire SIGLECF after MI. A) SIGLECF vs LY6G flow cytometry plots of cells from the indicated organs gated on viable CD45+CD11B+; B) proportion of SIGLECF+ neutrophils in the indicated organs in untouched mice, sham operated mice and MI-operated mice at 1 and 3 days after surgery.
    Figure Legend Snippet: Circulating, bone marrow and splenic neutrophils do not acquire SIGLECF after MI. A) SIGLECF vs LY6G flow cytometry plots of cells from the indicated organs gated on viable CD45+CD11B+; B) proportion of SIGLECF+ neutrophils in the indicated organs in untouched mice, sham operated mice and MI-operated mice at 1 and 3 days after surgery.

    Techniques Used: Flow Cytometry, Mouse Assay

    scRNA-seq of control and atherosclerotic aortas reveals two distinct neutrophil subsets. A) tSNE representation of single-cell RNA-seq gene expression data and clustering analysis of 2106 CD45 + cells isolated from control and atherosclerotic aortas from Ldlr −/− mice with identification of the major cell lineages; B) expression of Siglecf and Icam1 in cells corresponding to neutrophils projected onto the tSNE plot (for clarity, an expression cutoff has been applied; blue= transcript detected; grey=transcript not detected); C) experimental condition of origin of neutrophils projected onto the tSNE plot; D) violin plot showing log normalized expression of the indicated genes in the two neutrophil populations.
    Figure Legend Snippet: scRNA-seq of control and atherosclerotic aortas reveals two distinct neutrophil subsets. A) tSNE representation of single-cell RNA-seq gene expression data and clustering analysis of 2106 CD45 + cells isolated from control and atherosclerotic aortas from Ldlr −/− mice with identification of the major cell lineages; B) expression of Siglecf and Icam1 in cells corresponding to neutrophils projected onto the tSNE plot (for clarity, an expression cutoff has been applied; blue= transcript detected; grey=transcript not detected); C) experimental condition of origin of neutrophils projected onto the tSNE plot; D) violin plot showing log normalized expression of the indicated genes in the two neutrophil populations.

    Techniques Used: RNA Sequencing Assay, Expressing, Isolation, Mouse Assay

    Flow cytometry analysis of neutrophil susbets in the heart 5 days after myocardial infarction. A) Representative dot plot showing expression of SIGLECF on eosinophils (Eos, grey) and neutrophils (Neu, black); B) SSC/FSC profile of neutrophil subsets and eosinophils at 5 days after myocardial infarction; C) proportion of SIGLECF hi and SIGLECF low neutrophils among total LY6G+ neutrophils; surface level of D) ICAM1 and E) CD49d in neutrophils subsets; ***p
    Figure Legend Snippet: Flow cytometry analysis of neutrophil susbets in the heart 5 days after myocardial infarction. A) Representative dot plot showing expression of SIGLECF on eosinophils (Eos, grey) and neutrophils (Neu, black); B) SSC/FSC profile of neutrophil subsets and eosinophils at 5 days after myocardial infarction; C) proportion of SIGLECF hi and SIGLECF low neutrophils among total LY6G+ neutrophils; surface level of D) ICAM1 and E) CD49d in neutrophils subsets; ***p

    Techniques Used: Flow Cytometry, Expressing

    General data and scRNA-seq metrics. A) Proportion of CD11B + LY6G + neutrophils among total cardiac CD45+ leukocytes at 0 (control heart) and 1, 3, 5 and 7 days after myocardial infarction (MI); B) violin plot indicating the number of genes detected in each cell in the datasets analyzed in this report; C) percent of transcripts encoding ribosomal proteins in neutrophils according to their time point of origin; D) percent of transcripts encoding ribosomal proteins (name starting with Rps- or Rpl-) projected onto the tSNE plot of the global CD11B+ cells dataset shows higher proportion of these transcripts in non-neutrophil CD11B+ cells; E) hashtag antibody signal used to determine time point of origin of individual neutrophils (for clarity, minimum and maximum expression cutoffs have been applied); F) violin plot showing log normalized expression of characteristic tissue dissociation induced immediate early gene in neutrophil clusters.
    Figure Legend Snippet: General data and scRNA-seq metrics. A) Proportion of CD11B + LY6G + neutrophils among total cardiac CD45+ leukocytes at 0 (control heart) and 1, 3, 5 and 7 days after myocardial infarction (MI); B) violin plot indicating the number of genes detected in each cell in the datasets analyzed in this report; C) percent of transcripts encoding ribosomal proteins in neutrophils according to their time point of origin; D) percent of transcripts encoding ribosomal proteins (name starting with Rps- or Rpl-) projected onto the tSNE plot of the global CD11B+ cells dataset shows higher proportion of these transcripts in non-neutrophil CD11B+ cells; E) hashtag antibody signal used to determine time point of origin of individual neutrophils (for clarity, minimum and maximum expression cutoffs have been applied); F) violin plot showing log normalized expression of characteristic tissue dissociation induced immediate early gene in neutrophil clusters.

    Techniques Used: Expressing

    SIGLECF hi neutrophils populate the heart at Day 3 post-MI. A) Siglecf scaled expression in single cells projected onto the tSNE plot (splitted according to time point of origin) ; B) top: SIGLECF vs LY6G flow cytometry plots of cardiac cells gated on viable CD45 + CD11B + CD64 low at 1 and 3 days after MI; bottom: FSC-A/SSC-A signal in gated LY6G − SIGLECF hi eosinophils, LY6G + SIGLECF hi and LY6G + SIGLECF low neutrophils; C) neutrophils/mg, SIGLECF hi neutrophils/mg, and proportion of SIGLECF hi cells among LY6G + neutrophils in the heart at 1 and 3 days after MI; D) immunofluorescence staining for SIGLECF in cryosections of hearts in the infarcted area at 1 and 3 days after MI, x200, scale bar 100µm (isotype control staining was performed on day 3 post-MI hearts).
    Figure Legend Snippet: SIGLECF hi neutrophils populate the heart at Day 3 post-MI. A) Siglecf scaled expression in single cells projected onto the tSNE plot (splitted according to time point of origin) ; B) top: SIGLECF vs LY6G flow cytometry plots of cardiac cells gated on viable CD45 + CD11B + CD64 low at 1 and 3 days after MI; bottom: FSC-A/SSC-A signal in gated LY6G − SIGLECF hi eosinophils, LY6G + SIGLECF hi and LY6G + SIGLECF low neutrophils; C) neutrophils/mg, SIGLECF hi neutrophils/mg, and proportion of SIGLECF hi cells among LY6G + neutrophils in the heart at 1 and 3 days after MI; D) immunofluorescence staining for SIGLECF in cryosections of hearts in the infarcted area at 1 and 3 days after MI, x200, scale bar 100µm (isotype control staining was performed on day 3 post-MI hearts).

    Techniques Used: Expressing, Flow Cytometry, Immunofluorescence, Staining

    13) Product Images from "Levels of circulating endothelial cells are low in idiopathic pulmonary fibrosis and are further reduced by anti-fibrotic treatments"

    Article Title: Levels of circulating endothelial cells are low in idiopathic pulmonary fibrosis and are further reduced by anti-fibrotic treatments

    Journal: BMC Medicine

    doi: 10.1186/s12916-015-0515-0

    Gating strategy for the identification of circulating endothelial cells ( CEC ) and endothelial progenitor cells ( EPC ). Debris, monocytes and dead cells were excluded by the use of an electronic gate and the dump channel, containing cells identified by mAbs against CD14 and a viability marker, i.e. LIVE/DEAD. CEC and EPC were identified on the basis of the expression of CD34, CD45 and CD133: CEC were defined as CD45dim, CD34+ and CD133− while EPC were defined as CD45−, CD34+ and CD133+. The expression of CD309 (VEGFR-2, KDR) was detected among EPC and CEC. FSC forward scatter, SSC side scatter
    Figure Legend Snippet: Gating strategy for the identification of circulating endothelial cells ( CEC ) and endothelial progenitor cells ( EPC ). Debris, monocytes and dead cells were excluded by the use of an electronic gate and the dump channel, containing cells identified by mAbs against CD14 and a viability marker, i.e. LIVE/DEAD. CEC and EPC were identified on the basis of the expression of CD34, CD45 and CD133: CEC were defined as CD45dim, CD34+ and CD133− while EPC were defined as CD45−, CD34+ and CD133+. The expression of CD309 (VEGFR-2, KDR) was detected among EPC and CEC. FSC forward scatter, SSC side scatter

    Techniques Used: Capillary Electrochromatography, Marker, Expressing

    Gating strategy for the identification of circulating collagen I+ cells, i.e. fibrocytes. Debris, B cells, monocytes and dead cells were removed by an electronic gate as described in the legend to Fig. 1 . Circulating fibrocytes were identified as CD34+, CD45+ and collagen I+. The expression of CXCR4 was then evaluated among circulating fibrocytes. Lower panels, referred to cultured human fibroblasts, represents a positive control of collagen I staining ( > 95 % cells were positive). FSC forward scatter, SSC side scatter
    Figure Legend Snippet: Gating strategy for the identification of circulating collagen I+ cells, i.e. fibrocytes. Debris, B cells, monocytes and dead cells were removed by an electronic gate as described in the legend to Fig. 1 . Circulating fibrocytes were identified as CD34+, CD45+ and collagen I+. The expression of CXCR4 was then evaluated among circulating fibrocytes. Lower panels, referred to cultured human fibroblasts, represents a positive control of collagen I staining ( > 95 % cells were positive). FSC forward scatter, SSC side scatter

    Techniques Used: Expressing, Cell Culture, Positive Control, Staining

    14) Product Images from "Real-Time Detection of Tumor Cells during Capture on a Filter Element Significantly Enhancing Detection Rate"

    Article Title: Real-Time Detection of Tumor Cells during Capture on a Filter Element Significantly Enhancing Detection Rate

    Journal: Biosensors

    doi: 10.3390/bios11090312

    Leukocytes stained with anti-CD45-PE (orange) and spiked HCT116 cells stained with anti-EpCAM-FITC (green) captured on the TEM-grid. Detected by fluorescence microscopy at 50-fold magnification and 200 ms illumination time. The images were recorded separately in the respective channel and overlaid afterwards.
    Figure Legend Snippet: Leukocytes stained with anti-CD45-PE (orange) and spiked HCT116 cells stained with anti-EpCAM-FITC (green) captured on the TEM-grid. Detected by fluorescence microscopy at 50-fold magnification and 200 ms illumination time. The images were recorded separately in the respective channel and overlaid afterwards.

    Techniques Used: Staining, Transmission Electron Microscopy, Fluorescence, Microscopy

    15) Product Images from "IL-37 Gene Modification Enhances the Protective Effects of Mesenchymal Stromal Cells on Intestinal Ischemia Reperfusion Injury"

    Article Title: IL-37 Gene Modification Enhances the Protective Effects of Mesenchymal Stromal Cells on Intestinal Ischemia Reperfusion Injury

    Journal: Stem Cells International

    doi: 10.1155/2020/8883636

    IL-37-MSCs and MSCs could migrate to the injured tissue. The morphology of MSCs. (a) Passages 0, 1, 2, and 3 of MSCs. (b) FACS analysis of MSC surface marker, surface expressions of CD29, CD45, CD79a, and CD90 were detected. (c) The IL-37 and GFP proteins were detected in IL-37-MSCs and MSCs. (d) IL-37-MSC GFP-positive rate was calculated; positive rate was above 99.8% which met our needs. (e) IL-37-MSC-treated and MSC-treated intestine exhibited significant GFP fluorescence while the sham group did not, which suggested that IL-37-MSCs and MSCs could migrate to the injured tissue.
    Figure Legend Snippet: IL-37-MSCs and MSCs could migrate to the injured tissue. The morphology of MSCs. (a) Passages 0, 1, 2, and 3 of MSCs. (b) FACS analysis of MSC surface marker, surface expressions of CD29, CD45, CD79a, and CD90 were detected. (c) The IL-37 and GFP proteins were detected in IL-37-MSCs and MSCs. (d) IL-37-MSC GFP-positive rate was calculated; positive rate was above 99.8% which met our needs. (e) IL-37-MSC-treated and MSC-treated intestine exhibited significant GFP fluorescence while the sham group did not, which suggested that IL-37-MSCs and MSCs could migrate to the injured tissue.

    Techniques Used: FACS, Marker, Fluorescence

    16) Product Images from "Levels of circulating endothelial cells are low in idiopathic pulmonary fibrosis and are further reduced by anti-fibrotic treatments"

    Article Title: Levels of circulating endothelial cells are low in idiopathic pulmonary fibrosis and are further reduced by anti-fibrotic treatments

    Journal: BMC Medicine

    doi: 10.1186/s12916-015-0515-0

    Gating strategy for the identification of circulating endothelial cells ( CEC ) and endothelial progenitor cells ( EPC ). Debris, monocytes and dead cells were excluded by the use of an electronic gate and the dump channel, containing cells identified by mAbs against CD14 and a viability marker, i.e. LIVE/DEAD. CEC and EPC were identified on the basis of the expression of CD34, CD45 and CD133: CEC were defined as CD45dim, CD34+ and CD133− while EPC were defined as CD45−, CD34+ and CD133+. The expression of CD309 (VEGFR-2, KDR) was detected among EPC and CEC. FSC forward scatter, SSC side scatter
    Figure Legend Snippet: Gating strategy for the identification of circulating endothelial cells ( CEC ) and endothelial progenitor cells ( EPC ). Debris, monocytes and dead cells were excluded by the use of an electronic gate and the dump channel, containing cells identified by mAbs against CD14 and a viability marker, i.e. LIVE/DEAD. CEC and EPC were identified on the basis of the expression of CD34, CD45 and CD133: CEC were defined as CD45dim, CD34+ and CD133− while EPC were defined as CD45−, CD34+ and CD133+. The expression of CD309 (VEGFR-2, KDR) was detected among EPC and CEC. FSC forward scatter, SSC side scatter

    Techniques Used: Capillary Electrochromatography, Marker, Expressing

    Gating strategy for the identification of circulating collagen I+ cells, i.e. fibrocytes. Debris, B cells, monocytes and dead cells were removed by an electronic gate as described in the legend to Fig. 1 . Circulating fibrocytes were identified as CD34+, CD45+ and collagen I+. The expression of CXCR4 was then evaluated among circulating fibrocytes. Lower panels, referred to cultured human fibroblasts, represents a positive control of collagen I staining ( > 95 % cells were positive). FSC forward scatter, SSC side scatter
    Figure Legend Snippet: Gating strategy for the identification of circulating collagen I+ cells, i.e. fibrocytes. Debris, B cells, monocytes and dead cells were removed by an electronic gate as described in the legend to Fig. 1 . Circulating fibrocytes were identified as CD34+, CD45+ and collagen I+. The expression of CXCR4 was then evaluated among circulating fibrocytes. Lower panels, referred to cultured human fibroblasts, represents a positive control of collagen I staining ( > 95 % cells were positive). FSC forward scatter, SSC side scatter

    Techniques Used: Expressing, Cell Culture, Positive Control, Staining

    17) Product Images from "Circulating Monocytes from Systemic Sclerosis Patients with Interstitial Lung Disease Show an Enhanced Profibrotic Phenotype"

    Article Title: Circulating Monocytes from Systemic Sclerosis Patients with Interstitial Lung Disease Show an Enhanced Profibrotic Phenotype

    Journal: Laboratory investigation; a journal of technical methods and pathology

    doi: 10.1038/labinvest.2010.73

    Subgroup analysis of peripheral blood monocyte subpopulations and fibrogenic mediators in young vs. aged controls. A : Staining with anti-CD45-PE vs. intracellular isotype control antibody used to set negative gates. B :CD45 + /pro-Col-1α + staining (CD45-PE vs. pro-Col-Iα) in young healthy control. C : Anti-CD45-PE vs intracellular isotype control antibody used to set negative gates. D : CD45+/pro-Col-1α + staining (CD45-PE vs. pro-Col-Iα) in aged healthy control. E : Fibrocyte percentages (left axis) and quantities (right axis) in young subjects (white bar), aged subjects (black bar) and SSc-ILD patients (slanted lines). F : Comparison of lymphocytes (dotted line), monocytes (checked bar) and CD45 + /pro-Col-Iα + cells (black bar) in (left to right) young, aged, and SSc-ILD subjects. G : CD14+ monocyte expression of CD163 after 48 hours in culture. H-O : Concentrations of the plasma cytokines IL-4 (H), IL-10 (I), IL-13 (J), IL-1 RA (K), MCP-1 (L), TNFα (M), CCL-18 (N) and M-CSF (O) in young controls (n = 10, white bar), aged controls ( n = 12, black bar) and patients with SSc-ILD (n = 11, slanted bar). All data are expressed as mean ± SEM. * p
    Figure Legend Snippet: Subgroup analysis of peripheral blood monocyte subpopulations and fibrogenic mediators in young vs. aged controls. A : Staining with anti-CD45-PE vs. intracellular isotype control antibody used to set negative gates. B :CD45 + /pro-Col-1α + staining (CD45-PE vs. pro-Col-Iα) in young healthy control. C : Anti-CD45-PE vs intracellular isotype control antibody used to set negative gates. D : CD45+/pro-Col-1α + staining (CD45-PE vs. pro-Col-Iα) in aged healthy control. E : Fibrocyte percentages (left axis) and quantities (right axis) in young subjects (white bar), aged subjects (black bar) and SSc-ILD patients (slanted lines). F : Comparison of lymphocytes (dotted line), monocytes (checked bar) and CD45 + /pro-Col-Iα + cells (black bar) in (left to right) young, aged, and SSc-ILD subjects. G : CD14+ monocyte expression of CD163 after 48 hours in culture. H-O : Concentrations of the plasma cytokines IL-4 (H), IL-10 (I), IL-13 (J), IL-1 RA (K), MCP-1 (L), TNFα (M), CCL-18 (N) and M-CSF (O) in young controls (n = 10, white bar), aged controls ( n = 12, black bar) and patients with SSc-ILD (n = 11, slanted bar). All data are expressed as mean ± SEM. * p

    Techniques Used: Staining, Expressing

    18) Product Images from "Cyclic Stretch Induces Vascular Smooth Muscle Cells to Secrete Connective Tissue Growth Factor and Promote Endothelial Progenitor Cell Differentiation and Angiogenesis"

    Article Title: Cyclic Stretch Induces Vascular Smooth Muscle Cells to Secrete Connective Tissue Growth Factor and Promote Endothelial Progenitor Cell Differentiation and Angiogenesis

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2020.606989

    Five percent cyclic stretch induces VSMC-derived CTGF secretion and promotes the differentiation of cocultured EPCs into ECs. (A) Schematic diagrams show EPC//VSMC coculture and the cyclic stretch system. (B) EPCs showed a spindle-shaped morphology after 8 days. Staining of FITC-UEA-lectin (green) and Dil-acLDL (red) revealed double-positive cells that were identified as EPCs (a). FACS analysis showed that EPCs were positive for the endothelial cell marker CD31 and hematopoietic stem cell markers CD34 and CD133, and they were negative for the leukocyte marker CD45. Controls (blue area) were overlaid on the histogram of each surface antigen (red areas) tested (b). (C) The mRNA levels of the EC markers CD31, vWF, and KDR showed no differences in EPCs among different cocultured conditions after 6 h ( n = 5). Monocultured EPCs under static were used as control groups, shown as the dotted line. (D) The mRNA levels of the EC markers CD31, vWF, and KDR were induced in EPCs cocultured with stretched VSMCs after 12 h ( n = 5). (E) EPCs cocultured with stretched VSMCs had an increased tube formation ability after 12 h ( n = 5). (F) QPCR results revealed that CTGF mRNA levels were increased at different time points in stretched VSMCs ( n = 5). (G) The level of CTGF secretion from stretched VSMCs was significantly elevated at 12 h ( n = 5). For quantitative analysis, five fields per plate were photographed, and tube lengths were measured using Image-Pro Plus software. Scale bar = 100 μm. Values are expressed as the mean ± SD . *, # P
    Figure Legend Snippet: Five percent cyclic stretch induces VSMC-derived CTGF secretion and promotes the differentiation of cocultured EPCs into ECs. (A) Schematic diagrams show EPC//VSMC coculture and the cyclic stretch system. (B) EPCs showed a spindle-shaped morphology after 8 days. Staining of FITC-UEA-lectin (green) and Dil-acLDL (red) revealed double-positive cells that were identified as EPCs (a). FACS analysis showed that EPCs were positive for the endothelial cell marker CD31 and hematopoietic stem cell markers CD34 and CD133, and they were negative for the leukocyte marker CD45. Controls (blue area) were overlaid on the histogram of each surface antigen (red areas) tested (b). (C) The mRNA levels of the EC markers CD31, vWF, and KDR showed no differences in EPCs among different cocultured conditions after 6 h ( n = 5). Monocultured EPCs under static were used as control groups, shown as the dotted line. (D) The mRNA levels of the EC markers CD31, vWF, and KDR were induced in EPCs cocultured with stretched VSMCs after 12 h ( n = 5). (E) EPCs cocultured with stretched VSMCs had an increased tube formation ability after 12 h ( n = 5). (F) QPCR results revealed that CTGF mRNA levels were increased at different time points in stretched VSMCs ( n = 5). (G) The level of CTGF secretion from stretched VSMCs was significantly elevated at 12 h ( n = 5). For quantitative analysis, five fields per plate were photographed, and tube lengths were measured using Image-Pro Plus software. Scale bar = 100 μm. Values are expressed as the mean ± SD . *, # P

    Techniques Used: Derivative Assay, Staining, FACS, Marker, Real-time Polymerase Chain Reaction, Software

    19) Product Images from "Advances in rare cell isolation: an optimization and evaluation study"

    Article Title: Advances in rare cell isolation: an optimization and evaluation study

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-016-1108-1

    Cell image analysis. Cells are recorded at 4 different appearances from left to right ; under brightfield, AO fluorescence, PE fluorescence and contrast enhanced (ImageJ manual brightness adjustment) PE fluorescence. The bars represent a length of 10 µm. a CD45 positive cell identification. The cell indicated by a white arrow shows low AO as well as ring shaped PE fluorescence. CD45 negative cell identification with normal ( b ) and high (c) AO fluorescence intensity without ring formation in the light wavelength above 550 nm. Residual fluorescence is pronounced ( b ) and at noise level ( c ), visible only after contrast enhancement. The fluorescence signal is ascribed to AO fluorescence only
    Figure Legend Snippet: Cell image analysis. Cells are recorded at 4 different appearances from left to right ; under brightfield, AO fluorescence, PE fluorescence and contrast enhanced (ImageJ manual brightness adjustment) PE fluorescence. The bars represent a length of 10 µm. a CD45 positive cell identification. The cell indicated by a white arrow shows low AO as well as ring shaped PE fluorescence. CD45 negative cell identification with normal ( b ) and high (c) AO fluorescence intensity without ring formation in the light wavelength above 550 nm. Residual fluorescence is pronounced ( b ) and at noise level ( c ), visible only after contrast enhancement. The fluorescence signal is ascribed to AO fluorescence only

    Techniques Used: Fluorescence

    20) Product Images from "Immunoprophylactic and immunotherapeutic control of hormone receptor-positive breast cancer"

    Article Title: Immunoprophylactic and immunotherapeutic control of hormone receptor-positive breast cancer

    Journal: Nature Communications

    doi: 10.1038/s41467-020-17644-0

    NAM-treated tumors exhibit improved antigen presentation and superior cytotoxic functions. a tSNE plots of untreated and NAM-treated TSA tumors. Number of cells in each of the main four populations is reported. b Differential expression of genes involved in immune regulation in CD45 + cells isolated from untreated vs NAM-treated TSA tumors. Results are mean ± SEM plus individual data points. Number of cells independently analyzed for each gene is reported in ( a ), p values (two-sided Wilcoxon test) are indicated. See also Supplementary Data 1 . c , d Percentage of IFNG + and IFNG + GZMB + amongst CD8 + T cells ( c ) and CD56 + NK cells ( d ) from peripheral blood mononuclear cells (PBMCs) of healthy donors subjected to non-specific activation overnight in the presence of the indicated concentrations of NAM. Results are means ± SEM plus individual data points. Number of biologically independent samples and p values (one way-ANOVA plus Fisher LSD, as compared to untreated cells) are reported. e Relative expression levels of Ifnb1 and Ccl2 in TSA cells cultured in control conditions or exposed to the indicated concentrations of NAM for 48 h. Results are means ± SEM plus individual data points. Number of biologically independent samples and p values (one way-ANOVA plus Fisher LSD, as compared to untreated cells) are reported.
    Figure Legend Snippet: NAM-treated tumors exhibit improved antigen presentation and superior cytotoxic functions. a tSNE plots of untreated and NAM-treated TSA tumors. Number of cells in each of the main four populations is reported. b Differential expression of genes involved in immune regulation in CD45 + cells isolated from untreated vs NAM-treated TSA tumors. Results are mean ± SEM plus individual data points. Number of cells independently analyzed for each gene is reported in ( a ), p values (two-sided Wilcoxon test) are indicated. See also Supplementary Data 1 . c , d Percentage of IFNG + and IFNG + GZMB + amongst CD8 + T cells ( c ) and CD56 + NK cells ( d ) from peripheral blood mononuclear cells (PBMCs) of healthy donors subjected to non-specific activation overnight in the presence of the indicated concentrations of NAM. Results are means ± SEM plus individual data points. Number of biologically independent samples and p values (one way-ANOVA plus Fisher LSD, as compared to untreated cells) are reported. e Relative expression levels of Ifnb1 and Ccl2 in TSA cells cultured in control conditions or exposed to the indicated concentrations of NAM for 48 h. Results are means ± SEM plus individual data points. Number of biologically independent samples and p values (one way-ANOVA plus Fisher LSD, as compared to untreated cells) are reported.

    Techniques Used: Expressing, Isolation, Activation Assay, Cell Culture

    21) Product Images from "Optical imaging of the peri-tumoral inflammatory response in breast cancer"

    Article Title: Optical imaging of the peri-tumoral inflammatory response in breast cancer

    Journal: Journal of Translational Medicine

    doi: 10.1186/1479-5876-7-94

    Immunofluorescence/confocal microscopy . Top row, left to right: CD45, DiD. Bottom row: DAPI, merged image. Confocal images are representative of the MMTV-PymT control mice injected with DiD-labeled monocytes. Images are at 10× magnification.
    Figure Legend Snippet: Immunofluorescence/confocal microscopy . Top row, left to right: CD45, DiD. Bottom row: DAPI, merged image. Confocal images are representative of the MMTV-PymT control mice injected with DiD-labeled monocytes. Images are at 10× magnification.

    Techniques Used: Immunofluorescence, Confocal Microscopy, Mouse Assay, Injection, Labeling

    22) Product Images from "Independent recruitment of Igh alleles in V(D)J recombination"

    Article Title: Independent recruitment of Igh alleles in V(D)J recombination

    Journal: Nature Communications

    doi: 10.1038/ncomms6623

    Direct test for a stable Igh epigenetic commitment in HSC that dictates which allele undergoes V H to DJ H rearrangement first in the B lineage. ( a ) The expression of FOXP3GFP in lymph node (LN)-derived regulatory T cells is bimodal in the non-clonal populations, and unimodal in mice reconstituted with a single HSC (top histograms), but the expression of the Igh alleles is balanced in the splenic B cells of both the non-clonal and single-cell-reconstituted mice (bottom contour plots). Top histograms represent GFP expression profiles of CD45.1 + CD4 + FOXP3 + -gated LN cells, and bottom contour plots show the CD45.1 + CD19 + -gated splenic B-cell surface IgM a /IgM b profiles of the same clonal and non-clonal samples. ( b ) Genomic quantification with qPCR of in vivo -differentiated B-cell clones. The retention of the V H – D H intergenic fragment in IgM a and IgM b -sorted splenic populations from each of the single-HSC clones—the same samples shown in a —is similar to each other, and in the same range of IgM + -sorted cells from non-clonal populations. The qPCR on DNA from IgM + cells purified from Jht +/− mice (that can only rearrange one of the Igh alleles) and Atm −/− mice (which have a higher frequency of V H DJ H /V H DJ H cells than the WT 3 22 ) produced the expected results. Error bars are the s.e.m. of three independent runs of the same samples in different input amounts. ( c ) The reduction in the amount of the V H – D H intergenic fragment from the fractions A and B of pro-B cells to immature B cells shows that the assay is reporting the changes in the structure of the Igh locus throughout development. ( d ) Comparison of the frequency of productive and non-productive V H DJ H rearrangements detected in the splenic IgM a and IgM b -sorted pools from clone no. 5.1_3.5 M ( P =1.00, Fisher’s exact test) and no.5.2_3.5 M ( P =0.55, Fisher’s exact test).
    Figure Legend Snippet: Direct test for a stable Igh epigenetic commitment in HSC that dictates which allele undergoes V H to DJ H rearrangement first in the B lineage. ( a ) The expression of FOXP3GFP in lymph node (LN)-derived regulatory T cells is bimodal in the non-clonal populations, and unimodal in mice reconstituted with a single HSC (top histograms), but the expression of the Igh alleles is balanced in the splenic B cells of both the non-clonal and single-cell-reconstituted mice (bottom contour plots). Top histograms represent GFP expression profiles of CD45.1 + CD4 + FOXP3 + -gated LN cells, and bottom contour plots show the CD45.1 + CD19 + -gated splenic B-cell surface IgM a /IgM b profiles of the same clonal and non-clonal samples. ( b ) Genomic quantification with qPCR of in vivo -differentiated B-cell clones. The retention of the V H – D H intergenic fragment in IgM a and IgM b -sorted splenic populations from each of the single-HSC clones—the same samples shown in a —is similar to each other, and in the same range of IgM + -sorted cells from non-clonal populations. The qPCR on DNA from IgM + cells purified from Jht +/− mice (that can only rearrange one of the Igh alleles) and Atm −/− mice (which have a higher frequency of V H DJ H /V H DJ H cells than the WT 3 22 ) produced the expected results. Error bars are the s.e.m. of three independent runs of the same samples in different input amounts. ( c ) The reduction in the amount of the V H – D H intergenic fragment from the fractions A and B of pro-B cells to immature B cells shows that the assay is reporting the changes in the structure of the Igh locus throughout development. ( d ) Comparison of the frequency of productive and non-productive V H DJ H rearrangements detected in the splenic IgM a and IgM b -sorted pools from clone no. 5.1_3.5 M ( P =1.00, Fisher’s exact test) and no.5.2_3.5 M ( P =0.55, Fisher’s exact test).

    Techniques Used: Expressing, Derivative Assay, Mouse Assay, Real-time Polymerase Chain Reaction, In Vivo, Clone Assay, Purification, Produced

    23) Product Images from "Influenza infection triggers disease in a genetic model of experimental autoimmune encephalomyelitis"

    Article Title: Influenza infection triggers disease in a genetic model of experimental autoimmune encephalomyelitis

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

    doi: 10.1073/pnas.1620415114

    Gating strategy for comparing percentages of leukocytes isolated from brain and blood. ( A and B ) The gating strategy used to determine the effect of infection on percentages of B-cells (CD45 hi CD19 + ), T-cells (CD45 hi CD3 + ), and monocyte/neutrophils (CD45 hi CD11b + ) isolated from the brain ( A ) and peripheral blood ( B ) is shown.
    Figure Legend Snippet: Gating strategy for comparing percentages of leukocytes isolated from brain and blood. ( A and B ) The gating strategy used to determine the effect of infection on percentages of B-cells (CD45 hi CD19 + ), T-cells (CD45 hi CD3 + ), and monocyte/neutrophils (CD45 hi CD11b + ) isolated from the brain ( A ) and peripheral blood ( B ) is shown.

    Techniques Used: Isolation, Infection

    Influenza inoculation increased T-cell surveillance of the brain. ( A–E ) Mice were inoculated with saline ( n = 12) or influenza ( n = 20). On days 2, 4, 8, and 16 p.i., CNS-IL were isolated and stained with antibodies specific for hematogenous cells (CD45 + ), T-cells (CD3 + ), and B-cells (CD19 + ). ( A ) Effect of infection on weights ( Left ) and total number of cells isolated from brain ( Right ). ( B ) Gating strategy for viable and single cells. ( C and D ) Representative flow cytometry plots, cell percentage, and numbers of CD45 + CD3 + T-cells ( C ) and CD45 + CD19 + B-cells ( D ) in saline-inoculated mice ( n = 3) and influenza-inoculated mice ( n = 5) at days 2, 4, 8, and 16 in the brain. Significance was determined by two-way ANOVA. Results are means ± SE; ** P
    Figure Legend Snippet: Influenza inoculation increased T-cell surveillance of the brain. ( A–E ) Mice were inoculated with saline ( n = 12) or influenza ( n = 20). On days 2, 4, 8, and 16 p.i., CNS-IL were isolated and stained with antibodies specific for hematogenous cells (CD45 + ), T-cells (CD3 + ), and B-cells (CD19 + ). ( A ) Effect of infection on weights ( Left ) and total number of cells isolated from brain ( Right ). ( B ) Gating strategy for viable and single cells. ( C and D ) Representative flow cytometry plots, cell percentage, and numbers of CD45 + CD3 + T-cells ( C ) and CD45 + CD19 + B-cells ( D ) in saline-inoculated mice ( n = 3) and influenza-inoculated mice ( n = 5) at days 2, 4, 8, and 16 in the brain. Significance was determined by two-way ANOVA. Results are means ± SE; ** P

    Techniques Used: Mouse Assay, Isolation, Staining, Infection, Flow Cytometry, Cytometry

    24) Product Images from "Distinct populations of crypt-associated fibroblasts act as signaling hubs to control colon homeostasis"

    Article Title: Distinct populations of crypt-associated fibroblasts act as signaling hubs to control colon homeostasis

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.3001032

    Unbiased analysis of murine colon landscape reveals complexity and heterogeneity of epithelial and mesenchymal cells. (A) Schematic representation of a colonic crypt with surface markers used to sort epithelial (green: EpCAM+, CD45−) and mesenchymal/non-epithelial cells (red: EpCAM−, CD45−). (B) Flow cytometry analysis using EpCAM and CD45 on single-cell suspension of colonic epithelial (left, raw data: S2 Data ) and mesenchymal cell isolation (right, raw data: S3 Data ) prior to the sort. (C) Left: three major groups (epithelial, stromal, and endothelial) clustering into 16 distinct cluster revealed by UMAP analysis. Right: Epithelial cells show higher proliferative activity (cells in S-G2M) than non-epithelial cells (UMAP, single cells are colored according to their cluster annotation (left) or cell cycle phase (right)). (D) Characterization of cluster identity using relative expression of marker genes. (Dot plot, size, and color of the dot represent the percentage of cells which express the transcript and the average expression level within a cluster, respectively). EE, enteroendocrine; IESC, intestinal epithelial stem cell; TA, transit-amplifying cell; UMAP, Uniform Manifold Approximation and Projection.
    Figure Legend Snippet: Unbiased analysis of murine colon landscape reveals complexity and heterogeneity of epithelial and mesenchymal cells. (A) Schematic representation of a colonic crypt with surface markers used to sort epithelial (green: EpCAM+, CD45−) and mesenchymal/non-epithelial cells (red: EpCAM−, CD45−). (B) Flow cytometry analysis using EpCAM and CD45 on single-cell suspension of colonic epithelial (left, raw data: S2 Data ) and mesenchymal cell isolation (right, raw data: S3 Data ) prior to the sort. (C) Left: three major groups (epithelial, stromal, and endothelial) clustering into 16 distinct cluster revealed by UMAP analysis. Right: Epithelial cells show higher proliferative activity (cells in S-G2M) than non-epithelial cells (UMAP, single cells are colored according to their cluster annotation (left) or cell cycle phase (right)). (D) Characterization of cluster identity using relative expression of marker genes. (Dot plot, size, and color of the dot represent the percentage of cells which express the transcript and the average expression level within a cluster, respectively). EE, enteroendocrine; IESC, intestinal epithelial stem cell; TA, transit-amplifying cell; UMAP, Uniform Manifold Approximation and Projection.

    Techniques Used: Flow Cytometry, Cell Isolation, Activity Assay, Expressing, Marker

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  • 99
    Thermo Fisher anti rat cd45
    Neutralizing CCL20 reduces leukocyte infiltration. a Flow cytometry of the spinal cord at 7 days post-SCI shows the <t>CD45</t> + cell of different groups. b The frequency of CD45 + cell indicates that leukocyte infiltration was significantly alleviated by CCL20 neutralization. + P
    Anti Rat Cd45, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher cd45 monoclonal antibody
    Morphology of serially transplanted cervical cancer PDXs. A) representative example of an H E stained cervical squamous cell carcinoma sample showing morphology of the tumour biopsy, primary, secondary and tertiary PDXs. B) Typical examples of negative staining for anti-human <t>CD45</t> staining (second column), and anti-mouse CD45 (third column). Insets show examples of CD45 positive staining in human cervix biopsies and mouse kidney Scale bars 50 μm.
    Cd45 Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cd45 monoclonal antibody/product/Thermo Fisher
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    Price from $9.99 to $1999.99
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    93
    Thermo Fisher cd4
    Fas and FasL are expressed on the surface of infiltrating cells in reovirus 1/L-induced ARDS and BOOP. CBA/J mice were i.n. inoculated with either 1 × 10 6 PFU (BOOP) or 1 × 10 7 PFU (ARDS) reovirus 1/L and cells recovered by BAL were analyzed for the coexpression of cell surface phenotype markers with surface expression of Fas and FasL. Infiltrating cells were first stained with either <t>anti-CD4,</t> anti-CD8, anti-Mac1, or anti-GR1. The gated positive cells were then analyzed for the expression of either Fas or FasL. A , Cells recovered from ARDS-induced mice on day 9 postinoculation. B , Cells recovered from BOOP-induced mice on day 7 postinoculation. The percentage of the total cells expressing either anti-CD4, anti-CD8, anti-Mac1, or anti-GR1 is shown in the histograms on the left. The percentage of the gated population expressing either Fas or FasL is shown in the histograms on the right. The results shown represent one of the three independent experiments demonstrating similar results.
    Cd4, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher cd45 pe
    A: Confocal microscopy image of a frozen section of a tibia isolated from PPR*Tg/GFP mice. Tibias of 6-week-old mice were dissected and processed as described in Materials and Methods . Sections were stained with anti-GFP polyclonal, <t>anti-CD45</t> monoclonal,
    Cd45 Pe, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cd45 pe/product/Thermo Fisher
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    Image Search Results


    Neutralizing CCL20 reduces leukocyte infiltration. a Flow cytometry of the spinal cord at 7 days post-SCI shows the CD45 + cell of different groups. b The frequency of CD45 + cell indicates that leukocyte infiltration was significantly alleviated by CCL20 neutralization. + P

    Journal: Journal of Neuroinflammation

    Article Title: C-C motif chemokine ligand 20 regulates neuroinflammation following spinal cord injury via Th17 cell recruitment

    doi: 10.1186/s12974-016-0630-7

    Figure Lengend Snippet: Neutralizing CCL20 reduces leukocyte infiltration. a Flow cytometry of the spinal cord at 7 days post-SCI shows the CD45 + cell of different groups. b The frequency of CD45 + cell indicates that leukocyte infiltration was significantly alleviated by CCL20 neutralization. + P

    Article Snippet: Lymphocytes for CD45 flow cytometry were stained with cell surface markers: anti-rat CD45 (0.125 μg, PE, eBioscience, USA) or rat IgG1 K isotype control (0.125 μg, PE, eBioscience).

    Techniques: Flow Cytometry, Cytometry, Neutralization

    CCL20 neutralization reduces leukocyte infiltration. a Immunostaining of the spinal cord sections at 7 days post-SCI shows various amounts of CD45-positive cells in the different groups. CD45-positive cells are stained green , while the nuclei are stained blue . The immunostained areas in the white box are magnified below. Scale bars are 50 and 100 μm, respectively. b The percentage of CD45-positive cells indicates that leukocyte infiltration, as well as neuroinflammation during secondary injury, was significantly alleviated by neutralizing CCL20. + P

    Journal: Journal of Neuroinflammation

    Article Title: C-C motif chemokine ligand 20 regulates neuroinflammation following spinal cord injury via Th17 cell recruitment

    doi: 10.1186/s12974-016-0630-7

    Figure Lengend Snippet: CCL20 neutralization reduces leukocyte infiltration. a Immunostaining of the spinal cord sections at 7 days post-SCI shows various amounts of CD45-positive cells in the different groups. CD45-positive cells are stained green , while the nuclei are stained blue . The immunostained areas in the white box are magnified below. Scale bars are 50 and 100 μm, respectively. b The percentage of CD45-positive cells indicates that leukocyte infiltration, as well as neuroinflammation during secondary injury, was significantly alleviated by neutralizing CCL20. + P

    Article Snippet: Lymphocytes for CD45 flow cytometry were stained with cell surface markers: anti-rat CD45 (0.125 μg, PE, eBioscience, USA) or rat IgG1 K isotype control (0.125 μg, PE, eBioscience).

    Techniques: Neutralization, Immunostaining, Staining

    Morphology of serially transplanted cervical cancer PDXs. A) representative example of an H E stained cervical squamous cell carcinoma sample showing morphology of the tumour biopsy, primary, secondary and tertiary PDXs. B) Typical examples of negative staining for anti-human CD45 staining (second column), and anti-mouse CD45 (third column). Insets show examples of CD45 positive staining in human cervix biopsies and mouse kidney Scale bars 50 μm.

    Journal: PLoS ONE

    Article Title: A patient derived xenograft model of cervical cancer and cervical dysplasia

    doi: 10.1371/journal.pone.0206539

    Figure Lengend Snippet: Morphology of serially transplanted cervical cancer PDXs. A) representative example of an H E stained cervical squamous cell carcinoma sample showing morphology of the tumour biopsy, primary, secondary and tertiary PDXs. B) Typical examples of negative staining for anti-human CD45 staining (second column), and anti-mouse CD45 (third column). Insets show examples of CD45 positive staining in human cervix biopsies and mouse kidney Scale bars 50 μm.

    Article Snippet: For immunofluorescence PE-conjugated rat anti-mouse CD45 (eBioscience 12-0451-82), was incubated at a concentration of 1:100 for 60 minutes.

    Techniques: Staining, Negative Staining

    Fas and FasL are expressed on the surface of infiltrating cells in reovirus 1/L-induced ARDS and BOOP. CBA/J mice were i.n. inoculated with either 1 × 10 6 PFU (BOOP) or 1 × 10 7 PFU (ARDS) reovirus 1/L and cells recovered by BAL were analyzed for the coexpression of cell surface phenotype markers with surface expression of Fas and FasL. Infiltrating cells were first stained with either anti-CD4, anti-CD8, anti-Mac1, or anti-GR1. The gated positive cells were then analyzed for the expression of either Fas or FasL. A , Cells recovered from ARDS-induced mice on day 9 postinoculation. B , Cells recovered from BOOP-induced mice on day 7 postinoculation. The percentage of the total cells expressing either anti-CD4, anti-CD8, anti-Mac1, or anti-GR1 is shown in the histograms on the left. The percentage of the gated population expressing either Fas or FasL is shown in the histograms on the right. The results shown represent one of the three independent experiments demonstrating similar results.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Differential Role of the Fas/Fas Ligand Apoptotic Pathway in Inflammation and Lung Fibrosis Associated with Reovirus 1/L-Induced Bronchiolitis Obliterans Organizing Pneumonia and Acute Respiratory Distress Syndrome 1

    doi: 10.4049/jimmunol.0901958

    Figure Lengend Snippet: Fas and FasL are expressed on the surface of infiltrating cells in reovirus 1/L-induced ARDS and BOOP. CBA/J mice were i.n. inoculated with either 1 × 10 6 PFU (BOOP) or 1 × 10 7 PFU (ARDS) reovirus 1/L and cells recovered by BAL were analyzed for the coexpression of cell surface phenotype markers with surface expression of Fas and FasL. Infiltrating cells were first stained with either anti-CD4, anti-CD8, anti-Mac1, or anti-GR1. The gated positive cells were then analyzed for the expression of either Fas or FasL. A , Cells recovered from ARDS-induced mice on day 9 postinoculation. B , Cells recovered from BOOP-induced mice on day 7 postinoculation. The percentage of the total cells expressing either anti-CD4, anti-CD8, anti-Mac1, or anti-GR1 is shown in the histograms on the left. The percentage of the gated population expressing either Fas or FasL is shown in the histograms on the right. The results shown represent one of the three independent experiments demonstrating similar results.

    Article Snippet: The following Abs were used in this analysis: CD4 (GK1.5, L3T4; R-PE-labeled; Caltag Laboratories), CD8a (53-6.7, PerCP-labeled; BD Pharmingen), CD11b/Mac-1 (M1/70, allophycocyanin-labeled; BD Pharmingen), Ly6G (RB6-8C5, Gr-1, allophycocyanin-labeled; BD Pharmingen), Fas polyclonal Ab (A20, FITC-labeled; Santa Cruz Biotechnology), and FasL (Kay10, PE-labeled; BD Pharmingen).

    Techniques: Crocin Bleaching Assay, Mouse Assay, Expressing, Staining

    A: Confocal microscopy image of a frozen section of a tibia isolated from PPR*Tg/GFP mice. Tibias of 6-week-old mice were dissected and processed as described in Materials and Methods . Sections were stained with anti-GFP polyclonal, anti-CD45 monoclonal,

    Journal: The American Journal of Pathology

    Article Title: A Novel Population of Cells Expressing Both Hematopoietic and Mesenchymal Markers Is Present in the Normal Adult Bone Marrow and Is Augmented in a Murine Model of Marrow Fibrosis

    doi: 10.1016/j.ajpath.2011.10.028

    Figure Lengend Snippet: A: Confocal microscopy image of a frozen section of a tibia isolated from PPR*Tg/GFP mice. Tibias of 6-week-old mice were dissected and processed as described in Materials and Methods . Sections were stained with anti-GFP polyclonal, anti-CD45 monoclonal,

    Article Snippet: BM cells isolated from WT/GFP mice, with or without PTH treatment, or PPR*Tg/GFP mice were incubated with CD45-PE or CD45-APC and CD11b-APC antibodies (eBioscience) and analyzed as previously described.

    Techniques: Confocal Microscopy, Isolation, Mouse Assay, Staining

    A – C: Identification and characterization of GFP + CD45 + cells in WT and PPR*Tg mice. BM cells were isolated as described in Materials and Methods from WT/GFP ( A ) and PPR*Tg/GFP ( B ) mice, and analyzed for the expression of GFP, CD45, and CD11b. Note

    Journal: The American Journal of Pathology

    Article Title: A Novel Population of Cells Expressing Both Hematopoietic and Mesenchymal Markers Is Present in the Normal Adult Bone Marrow and Is Augmented in a Murine Model of Marrow Fibrosis

    doi: 10.1016/j.ajpath.2011.10.028

    Figure Lengend Snippet: A – C: Identification and characterization of GFP + CD45 + cells in WT and PPR*Tg mice. BM cells were isolated as described in Materials and Methods from WT/GFP ( A ) and PPR*Tg/GFP ( B ) mice, and analyzed for the expression of GFP, CD45, and CD11b. Note

    Article Snippet: BM cells isolated from WT/GFP mice, with or without PTH treatment, or PPR*Tg/GFP mice were incubated with CD45-PE or CD45-APC and CD11b-APC antibodies (eBioscience) and analyzed as previously described.

    Techniques: Mouse Assay, Isolation, Expressing

    Identification and characterization of GFP + CD45 + cells in Col2.3GFP mice after PTH treatment. An endosteal cell fraction (fraction 2) was isolated from Col2.3/GFP (treated and untreated) mice. Cells were stained with anti-CD45 antibody and analyzed by

    Journal: The American Journal of Pathology

    Article Title: A Novel Population of Cells Expressing Both Hematopoietic and Mesenchymal Markers Is Present in the Normal Adult Bone Marrow and Is Augmented in a Murine Model of Marrow Fibrosis

    doi: 10.1016/j.ajpath.2011.10.028

    Figure Lengend Snippet: Identification and characterization of GFP + CD45 + cells in Col2.3GFP mice after PTH treatment. An endosteal cell fraction (fraction 2) was isolated from Col2.3/GFP (treated and untreated) mice. Cells were stained with anti-CD45 antibody and analyzed by

    Article Snippet: BM cells isolated from WT/GFP mice, with or without PTH treatment, or PPR*Tg/GFP mice were incubated with CD45-PE or CD45-APC and CD11b-APC antibodies (eBioscience) and analyzed as previously described.

    Techniques: Mouse Assay, Isolation, Staining

    Flow cytometry analysis of BM cells isolated from WT and PPR*Tg mice. BM cells were collected as described in Materials and Methods , and were then incubated with ScaI-PE-Cy5.5, CD45-APC, and CD31-APC antibodies. A: Representative analysis of BM cells

    Journal: The American Journal of Pathology

    Article Title: A Novel Population of Cells Expressing Both Hematopoietic and Mesenchymal Markers Is Present in the Normal Adult Bone Marrow and Is Augmented in a Murine Model of Marrow Fibrosis

    doi: 10.1016/j.ajpath.2011.10.028

    Figure Lengend Snippet: Flow cytometry analysis of BM cells isolated from WT and PPR*Tg mice. BM cells were collected as described in Materials and Methods , and were then incubated with ScaI-PE-Cy5.5, CD45-APC, and CD31-APC antibodies. A: Representative analysis of BM cells

    Article Snippet: BM cells isolated from WT/GFP mice, with or without PTH treatment, or PPR*Tg/GFP mice were incubated with CD45-PE or CD45-APC and CD11b-APC antibodies (eBioscience) and analyzed as previously described.

    Techniques: Flow Cytometry, Cytometry, Isolation, Mouse Assay, Incubation