anti-mouse cd127 pe cy7  (Thermo Fisher)

Journal: Cell reports

Article Title: Transcriptional programming mediated by the histone demethylase KDM5C regulates dendritic cell population heterogeneity and function

doi: 10.1016/j.celrep.2024.114506

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-mouse CD127 PE/Cy7 (clone A7R34) , eBioscience , 25-1271-82; RRID:AB_469649.

Techniques: Control, Blocking Assay, Virus, Recombinant, Protease Inhibitor, Lysis, Enzyme-linked Immunosorbent Assay, Staining, Cell Isolation, DNA Library Preparation, Purification, RNA HS Assay, Mutagenesis, Software

Journal: Cell reports

Article Title: Splicing factor deficits render hematopoietic stem and progenitor cells sensitive to STAT3 inhibition

doi: 10.1016/j.celrep.2022.111825

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rat anti-mouse CD127-PE-Cy7 , BD Biosciences , 560733, RRID:AB_1727424.

Techniques: Purification, Recombinant, Clone Assay, Software

Journal: Journal for Immunotherapy of Cancer

Article Title: Type 17 immunity promotes the exhaustion of CD8 + T cells in cancer

doi: 10.1136/jitc-2021-002603

Figure Lengend Snippet: Tumor-infiltrating Tc17 cells exhibit tissue resident memory-like phenotypes in mice and humans. (A–E) Lung CD8 + tumor-infiltrating lymphocytes (TILs) in CD4-depleted B16F10 tumor-bearing C57BL/6 mice were analyzed. (A) Representative contour plots of cell surface expression of CD103 and KLRG1 on Tc17 and Tc1 cells. (B) PerCP-Cy5.5-labeled anti-mouse CD8α Ab was intravenously (i.v.) injected into CD4-depleted B16F10 tumor-bearing mice 5 min before sacrifice for analysis. Representative contour plots of CD8 α-PerCP-Cy5.5 + cells among total CD8 + T cells in spleen and lung tumor are shown. (C) Representative histogram of CD127 expression and frequencies of CD127 + cells of Tc17 and Tc1 cells (n=4). (D) Representative histogram of Ki-67 expression and frequencies of Ki-67 + cells of Tc17 and Tc1 cells (n=7). (E) Frequencies of granzyme B(GzmB) + cells of Tc17 and Tc1 cells (n=4). (F) Cytotoxicity of in vitro generated gp100-specific Tc1 cells or Tc17 cells (effector cells) against gp100-pulsed TC-1 tumor cells (target cells). Frequencies of Caspase-3(Casp3) + among CTV-labeled target cells were analyzed 4 hours after co-culture with effector cells. (G–J) CD8 + TILs isolated from tumor tissues of patients with hepatocellular carcinoma (HCC) were analyzed by flow cytometry. (G) Representative contour plots of IL-17 and IFN-γ production by Tc17 and Tc1 cells in TILs of patients with HCC and frequencies of Tc17 and Tc1 cells among CD8 + TILs (n=11). (H) Cell surface expression of CD103 and KLRG1 on each CD8 + T cell subset. (I) Representative histogram of cell surface expression of CD127 on each CD8 + TIL subset and frequencies of CD127 hi cells (n=6). (J) Representative histogram of GzmB expression in each CD8 + TIL subset and frequencies of GzmB + cells in each CD8 + TIL subset (n=6). Data are representatives of at least two independent experiments for (A–F) and (H–J) and combined results of two independent experiments for (G). Data are shown as mean±SD for (C-E, G, I, J) and mean±SEM for (F). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: For mouse sample flow cytometry analysis, lymphoid cells were obtained and stained with PerCP-Cy5.5 or PE-Cy7 anti-mouse CD8α (53-6.7; BioLegend), BUV395 or BUV737 anti-mouse CD8α (53-6.7; BD Biosciences), PerCP-Cy5.5 or APC anti-mouse CD4 (GK1.5; BioLegend), BV510 or PE-Cy7 anti-mouse CD4 (RM4-5; BioLegend), eFluor450 or PE-Cy7 anti-mouse CD90.2 (53-2.1; BioLegend), PerCP-Cy5.5 anti-mouse CD3ε (145-2 C11; BioLegend), BUV395 anti-mouse CD3ε (145-2 C11; BD Biosciences), Pacific Blue anti-mouse TCRβ (H57-597; BioLegend), APC anti-mouse γδTCR (GL-3; eBioscience), PE mouse CD1d tetramer (NIH Tetramer Core facility), APC anti-mouse/human KLRG-1 (2F1/KLRG1; BioLegend), APC-Cy7, FITC, or PE anti-mouse CD103 (2E7; BioLegend), Alexa Fluor 488, PerCP-Cy5.5 or Pacific Blue anti-mouse CD45.1 (A20; BioLegend), PerCP-Cy5.5, Pacific Blue, or BV510 anti-mouse CD45.2 (104; BioLegend), PE-Cy7 anti-mouse PD1 (RMP 1-30; BioLegend), PE or APC anti-mouse Tim3 (RMT3-23; eBioscience), APC or PE-Cy7 anti-mouse/human CD44 (IM7; BioLegend), Alexa Fluor 488 or PerCP-Cy5.5 anti-mouse CD62L (MEL-14; BioLegend), and PE-Cy7 or APC anti-mouse CD127 (A7R34; eBioscience).

Techniques: Expressing, Labeling, Injection, In Vitro, Generated, Co-Culture Assay, Isolation, Flow Cytometry

Journal: iScience

Article Title: CD8 memory precursor cell generation is a continuous process

doi: 10.1016/j.isci.2022.104927

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Article Snippet: PE-Cy7 anti-mouse CD127 (clone A7R34) , eBiosciences , Cat#25-1271-82; RRID: AB_469649.

Techniques: Virus, Modification, Western Blot, Recombinant, BrdU Staining, Flow Cytometry, Software

Journal: Nature Communications

Article Title: A DAP5/eIF3d alternate mRNA translation mechanism promotes differentiation and immune suppression by human regulatory T cells

doi: 10.1038/s41467-021-27087-w

Figure Lengend Snippet: a Representative parental populations of CD24 + CD127 −/dim CD25 + cells (representative of three donors). Human naive CD4 + lymphocytes were activated at d 1 and d 7 by αCD3, soluble αCD28, and 150 U/ml IL-2 and cultured for 12 d. Treatments of 20 nM RAD001, 2 ng/ml activated TGF-beta, RAD001+TGF-beta or DMSO (untreated) were added during activation and subsequent expansions as needed. IL-2 was refreshed every 2–3 d. After 12 d, cells were harvested, treated cells enriched for Treg cells by magnetic-labeling recovery. Sorted cells were stained and analyzed by flow cytometry. Total lymphocytes were gated for doublet exclusion, live CD4 + T cells, and CD25 + CD127 − cells as shown. b TGF-beta or RAD001 treatments increase the percentage of CD25 + CD127 − in CD4 + population. Quantitation of CD4 + CD127 dim/− CD25 + population for each treatment shown from a representative data of three donors. c TGF-beta treatment increases the percentage of FOXP3 + CD25 + cells in CD4 + population, shown from representative data of three donors. FMO-CD127 and FMO-FOXP3 were used as references for gating. d Lymphocytes from six different donors were cultured, treated, and stained as described in a . Percentages of FOXP3 + CD25 + cells for each donor were normalized to its untreated control and plotted cumulatively. e Number of CD25 + on CD4 + cells is unchanged by treatments. Samples from d were analyzed for percentage of overall CD25 + cells and for CD25 intensity (MFI, median fluorescence intensity of CD25-labeling fluorophore). Six independent donors per group were tested. f TGF-beta and RAD001+TGF-beta treatments increase numbers and intensity of expression of FOXP3 + on CD4 + T cells (MFI of FOXP3-labeling fluorophore). g iTreg cells were generated as in a , enriched for CD4 + CD127 dim/ − CD25 + cells by sorting, responder cells (autologous PBMCs) labeled with CFSE and co-cultured with labeled responders at different ratios with overnight activation. Suppression ability of treated and untreated cells for all ratios shown, expressed as 1-[division index of tested cells/average division index of responders alone]. Division indices obtained from the FlowJo Proliferation Platform of tests on three independent donors per test condition. a – g P values determined by statistical analysis using two-way ANOVA tests with Dunnett post-ANOVA test determination with SEM shown. Source data are provided as a Source Data file.

Article Snippet: In all, 1–2 × 10 5 cells were incubated with Blue LIVE/DEAD Fixable Dead Cell Stain kit (Life Technologies, Cat. # L-23105) or Zombie Aqua Fixable Viability Kit (BioLegend, Cat. # 423102) in 1× PBS, and then stained with surface antibodies diluted in Stain Buffer (BD Pharmingen, Cat. #554656): FITC mouse anti-human CD4 (BD Pharmingen, Cat. #555346), APC mouse anti-human CD25 (BD Pharmingen, Cat. #555434), PE-Cy7 mouse anti-human CD127 (Invitrogen BD Pharmingen, Cat. #5 25-1278-42), PE mouse anti-human CD101 (BioLegend, Cat. #331012), Brilliant Violet 421 mouse anti-human CD103 (BioLegend, Cat. #350213) and PE mouse anti-human GITR (BioLegend, Cat. #371203).

Techniques: Cell Culture, Activation Assay, Labeling, Staining, Flow Cytometry, Quantitation Assay, Fluorescence, Expressing, Generated

Journal: Nature Communications

Article Title: A DAP5/eIF3d alternate mRNA translation mechanism promotes differentiation and immune suppression by human regulatory T cells

doi: 10.1038/s41467-021-27087-w

Figure Lengend Snippet: Human naive CD4 + T cells were cultured and induced to differentiate to iTreg cells, then analyzed by flow cytometry as described in the legend to Fig. . a RAD001+TGF-beta-induced CD4 + CD127 dim/− CD25 + FOXP3 + iTreg cells that express high levels of GITR, CD101, CD103, TGF-beta, and IL-10. Quantitation was derived from three independent donors. P values were determined by statistical analysis using unpaired t test, with mean and SEM shown. b Representative CD4 + CD127 dim/− CD25 + FOXP3 + cell population from three studies expressing CD101, +/− TGF-beta, +/− RAD001 treatment. Percentages normalized to untreated control for each donor. FMO-CD127 and FMO-FOXP3 were used as references for gating. c Fold increase in CD101 and percentage of the CD4 + CD127 dim/− CD25 + FOXP3 + population obtained from three independent donor iTreg cells in b , normalized to each donor untreated control. d Representative flow cytometry of three independent donors of the CD4 + CD127 dim/− CD25 + FOXP3 + cell population expressing CD103, +/− TGF-beta, +/− RAD001 treatment. Percentages normalized to untreated control for each donor. FMO-CD127 and FMO-FOXP3 were used as a reference for gating. e Fold increase in CD103 and percentage of the CD4 + CD127 dim/− CD25 + FOXP3 + population from three donors in d normalized to each donor untreated control and plotted. P values were determined for c – e by two-way ANOVA test with Dunnett post-ANOVA test determination, with mean values and SEM shown. Source data are provided as a Source Data file.

Article Snippet: In all, 1–2 × 10 5 cells were incubated with Blue LIVE/DEAD Fixable Dead Cell Stain kit (Life Technologies, Cat. # L-23105) or Zombie Aqua Fixable Viability Kit (BioLegend, Cat. # 423102) in 1× PBS, and then stained with surface antibodies diluted in Stain Buffer (BD Pharmingen, Cat. #554656): FITC mouse anti-human CD4 (BD Pharmingen, Cat. #555346), APC mouse anti-human CD25 (BD Pharmingen, Cat. #555434), PE-Cy7 mouse anti-human CD127 (Invitrogen BD Pharmingen, Cat. #5 25-1278-42), PE mouse anti-human CD101 (BioLegend, Cat. #331012), Brilliant Violet 421 mouse anti-human CD103 (BioLegend, Cat. #350213) and PE mouse anti-human GITR (BioLegend, Cat. #371203).

Techniques: Cell Culture, Flow Cytometry, Quantitation Assay, Derivative Assay, Expressing

Journal: Nature Communications

Article Title: A DAP5/eIF3d alternate mRNA translation mechanism promotes differentiation and immune suppression by human regulatory T cells

doi: 10.1038/s41467-021-27087-w

Figure Lengend Snippet: a Schema for silencing DAP5 in activated CD4 + T cells during differentiation. Human naive CD4 + T cells from two different donors were isolated from PBMCs as described in Fig. legend. Cells were treated with 1 μM of Accell SMARTpool siRNAs targeting DAP5 or a non-targeting siRNA pool in serum-free X-Vivo 15 media containing 1% GlutaMAX for 24 h, activated with IL-2 and differentiated with TGF-beta and RAD001 to induce iTreg cells and maintained as described in Methods. Accell SMARTpool siRNAs were re-added to culture medium on d 3 and 6 after activation and cells submitted to flow cytometry on d 13. Studies included a GFP-siRNA non-targeting control to measure uptake efficiency by flow cytometry. b Levels of GFP uptake measured by flow cytometry in negative control (no GFP), IL-2 activated but otherwise untreated, and RAD001+TGF-beta treated (R+T) CD4 + T cells. Representative plot of two independent studies. c Equal protein amounts of cell lysates from two donors obtained as in a were pooled and subjected to immunoblot analysis. Samples correspond to untreated and RAD001 + TGF-beta treated cells. DAP5 protein levels were reduced by 70–80% with silencing. Immunoblots are representative of two independent experiments. d Human CD4 + T cells treated as in a were subjected to flow cytometry for differentiated iTreg cells (CD4 + CD127 dim/– CD25 + FOXP3 + GITR + ) determined on d 13. Representative flow plots of two independent experiments shown indicating a 51% reduction in differentiated iTreg cells to baseline levels of Treg cells isolated from PBMCs prior to differentiation. e iTreg cells were tested for viability by Trypan Blue exclusion assay. P values determined using Fisher’s exact test with means and SEM shown. There is no statistically significant difference in viability between pairs of non-silenced and DAP5 silenced matched conditions. f Percentages of CD4 + CD127 dim/− CD25 + FOXP3 + GITR + cells for each of two donors were normalized to its untreated control and plotted cumulatively for IL-2 activated but otherwise untreated, and RAD001+TGF-beta treated (R+T) CD4 + T cells. Source data are provided as a Source Data file.

Article Snippet: In all, 1–2 × 10 5 cells were incubated with Blue LIVE/DEAD Fixable Dead Cell Stain kit (Life Technologies, Cat. # L-23105) or Zombie Aqua Fixable Viability Kit (BioLegend, Cat. # 423102) in 1× PBS, and then stained with surface antibodies diluted in Stain Buffer (BD Pharmingen, Cat. #554656): FITC mouse anti-human CD4 (BD Pharmingen, Cat. #555346), APC mouse anti-human CD25 (BD Pharmingen, Cat. #555434), PE-Cy7 mouse anti-human CD127 (Invitrogen BD Pharmingen, Cat. #5 25-1278-42), PE mouse anti-human CD101 (BioLegend, Cat. #331012), Brilliant Violet 421 mouse anti-human CD103 (BioLegend, Cat. #350213) and PE mouse anti-human GITR (BioLegend, Cat. #371203).

Techniques: Isolation, Activation Assay, Flow Cytometry, Negative Control, Western Blot, Trypan Blue Exclusion Assay

Journal: Cell reports

Article Title: A stromal progenitor and ILC2 niche promotes muscle eosinophilia and fibrosis-associated gene expression

doi: 10.1016/j.celrep.2021.108997

Figure Lengend Snippet: (A) Gating strategy used to interrogate IL-5 (Red5)-expressing cells in mdx muscle. (B) Quantification of muscle IL-5 + cells using flow cytometry. n = 7–9. (C) Percent of stroma (CD45 − ), Lin + (Lin + CD4 − ), CD4 + (Lin + CD4 + ), and ILC2s (Lin − Thy1 + KLRG1 + CD127 + ) that express IL-5 in mdx muscle. n = 9. Statistics are compared to CD45 − . (D) Representative histograms showing the expression of common ILC2 markers on muscle CD45 + Thy1 + Lin − cells in WT and mdx mice. Iso, isotype control. n = 5–6. (E) Gating strategy used to interrogate muscle ILCs. (F–H) The number of muscle CD45 + Thy1 + NK1.1 + (F), CD45 + Thy1 + Lin − KLRG1 + (G), and CD45 + Thy1 + Lin − RORγt + (H) cells. n = 4. (I and J) The frequency (I) and number (J) of IL-13 + muscle ILC2s. (K and L) Representative histogram (K) and the average geometric mean fluorescence intensity (MFI) (L) of IL-13 expression in CD45 + Thy1 + Lin − KLRG1 + muscle ILC2s. n = 3–4 (I–L). 4-wk-old mice were analyzed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using an unpaired Welch’s t test (B, F–H, I, J, and L) or one-way ANOVA with Bonferroni correction (C).

Article Snippet: Anti-mouse CD127 (A7R34) PE-Cy7 , eBioscience , Cat #25–1271-82.

Techniques: Expressing, Flow Cytometry, Control, Fluorescence

Journal: Cell reports

Article Title: A stromal progenitor and ILC2 niche promotes muscle eosinophilia and fibrosis-associated gene expression

doi: 10.1016/j.celrep.2021.108997

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Article Snippet: Anti-mouse CD127 (A7R34) PE-Cy7 , eBioscience , Cat #25–1271-82.

Techniques: Recombinant, Saline, Blocking Assay, Plasmid Preparation, Lysis, cDNA Synthesis, Staining, Avidin-Biotin Assay, Software