pe cy7 anti human cd127 antibody  (Thermo Fisher)

Journal: STAR Protocols

Article Title: Protocol for in vitro generation of innate lymphoid cells from human embryonic tissues

doi: 10.1016/j.xpro.2024.103525

Figure Lengend Snippet: Antibody cocktail for lymphoid progenitors

Article Snippet: Anti-human CD127 PE-Cy7 clone: eBioRDR5 (dilution: 1:100) , Invitrogen , Cat#25-1278-42; RRID: AB_1659672.

Techniques:

Journal: STAR Protocols

Article Title: Protocol for in vitro generation of innate lymphoid cells from human embryonic tissues

doi: 10.1016/j.xpro.2024.103525

Figure Lengend Snippet: Representative flow cytometry plots for identifying lymphoid progenitors (CD45 + CD34 + CD127 + ) in YS (top) and EP (bottom)

Article Snippet: Anti-human CD127 PE-Cy7 clone: eBioRDR5 (dilution: 1:100) , Invitrogen , Cat#25-1278-42; RRID: AB_1659672.

Techniques: Flow Cytometry

Journal: STAR Protocols

Article Title: Protocol for in vitro generation of innate lymphoid cells from human embryonic tissues

doi: 10.1016/j.xpro.2024.103525

Figure Lengend Snippet: Antibody cocktail for cultured phenotype ILC subsets

Article Snippet: Anti-human CD127 PE-Cy7 clone: eBioRDR5 (dilution: 1:100) , Invitrogen , Cat#25-1278-42; RRID: AB_1659672.

Techniques: Cell Culture

Journal: STAR Protocols

Article Title: Protocol for in vitro generation of innate lymphoid cells from human embryonic tissues

doi: 10.1016/j.xpro.2024.103525

Figure Lengend Snippet: Antibody cocktail for surface markers of ILC subsets

Article Snippet: Anti-human CD127 PE-Cy7 clone: eBioRDR5 (dilution: 1:100) , Invitrogen , Cat#25-1278-42; RRID: AB_1659672.

Techniques:

Journal: STAR Protocols

Article Title: Protocol for in vitro generation of innate lymphoid cells from human embryonic tissues

doi: 10.1016/j.xpro.2024.103525

Figure Lengend Snippet: Antibody cocktail for intracellular transcription factors of ILC subsets

Article Snippet: Anti-human CD127 PE-Cy7 clone: eBioRDR5 (dilution: 1:100) , Invitrogen , Cat#25-1278-42; RRID: AB_1659672.

Techniques:

Journal: STAR Protocols

Article Title: Protocol for in vitro generation of innate lymphoid cells from human embryonic tissues

doi: 10.1016/j.xpro.2024.103525

Figure Lengend Snippet: Antibody cocktail for surface markers of cultured NK cells

Article Snippet: Anti-human CD127 PE-Cy7 clone: eBioRDR5 (dilution: 1:100) , Invitrogen , Cat#25-1278-42; RRID: AB_1659672.

Techniques: Cell Culture

Journal: STAR Protocols

Article Title: Protocol for in vitro generation of innate lymphoid cells from human embryonic tissues

doi: 10.1016/j.xpro.2024.103525

Figure Lengend Snippet:

Article Snippet: Anti-human CD127 PE-Cy7 clone: eBioRDR5 (dilution: 1:100) , Invitrogen , Cat#25-1278-42; RRID: AB_1659672.

Techniques: Recombinant, Saline, Staining, Blocking Assay, Low Protein Binding, Cell Stimulation, Cell Culture, Software, Cell Counting

Journal: Heliyon

Article Title: Dysregulated T-cell homeostasis and decreased CD30 + Treg proliferating in aplastic anemia

doi: 10.1016/j.heliyon.2024.e35775

Figure Lengend Snippet: Dysregulated homeostasis of naïve/memory CD4 + T cells and increased the differentiation of TH1, TH2, and TH17. (A). The scores of Naïve/memory, TH1, TH2, and TH17 gene sets in BM or PB CD4 + T cells of AA patients and HDs. (B) (i) Naïve/memory score in BM CD4 + T cells of AA patients and HDs (GSE181989 and E-MTAB-9969). The information on patients with AA and HDs can be obtained from Hu Tonglin et al. . (B) (ii). Changes of naïve/memory, TH1, TH2, and TH17 scores in PB or BM CD4 + T cells of AA patients were monitored during immunosuppressive therapy (IST) using dataset E-MTAB-9969. Patient AA-4, a 52-year-old female, exhibited a somatic STAT1 mutation P.Y640F in CD8 + T cells. Patient AA-3, a 58-year-old female, presented with sAA following two years of macrocytosis and mild thrombocytopenia, and displayed somatic mutations of KRAS , NFATC2 , PTPN22 , and TNFAIP3 . The treatment response information can be referenced from Sofie Lundgren et al. . (C). The branched trajectory of CD4 + T cells was organized by individuals, with each dot representing a single cell. (D). Expression maps display log-normalized expression of key functional genes, including STAT4 , STAT1 , IL4R , STAT6 , RORC , and IL6R , in the differentiation branches of naïve CD4 + T into TH1/TH2/TH17 cells. Data is represented as log-normalized expressions With yellow indicating high expression and dark blue indicating low expression. (E). Dot plots show the expression levels of CCR4 , CCR6 , KLRB1 , and IL7R in CD4 + T cells of AA patients and HD. (F). Representative flow cytometry dot plots illustrate the expression of CCR4 and CCR6 in CD4 + T cells from PB samples of AA patients and HDs. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The nonspecific binding of the immunoglobulin to the Fc receptors of PBMCs and BMMCs was blocked by FcR Blocking Reagent (No. 130–0590901, Miltenyi Biotec), and stained with the following antibodies: FITC anti-human CD8a antibody (RPA-T8, No. 301050, BioLegend), APC-Cy7 anti-human CD4 antibody (A161A1, No. 357415, BioLegend), PE anti-human CCR4 (CD194) antibody (L291H4, No. 359412, BioLegend), APC anti-human CCR6 (CD196) antibody (G034E3, No. 353416, BioLegend), Biotin anti-human CD161 antibody (HP-3G10, No. 339932, BioLegend), APC anti-human CD30 antibody (BY88, No. 333910, BioLegend), Percp-Cy5.5 anti-human CD25 antibody (BC96, No. 302626, BioLegend), and PE-Cy7 anti-human CD127 antibody (A7R34, No. 25-1271-82, eBioscience), combined with APC-Cy7 Streptavidin (No. 405208, BioLegend), and PE/Cyanine7 Streptavidin (No. 405206, BioLegend).

Techniques: Mutagenesis, Expressing, Functional Assay, Flow Cytometry

Journal: Heliyon

Article Title: Dysregulated T-cell homeostasis and decreased CD30 + Treg proliferating in aplastic anemia

doi: 10.1016/j.heliyon.2024.e35775

Figure Lengend Snippet: Dysregulated naïve/memory CD8 + T cell homeostasis. (A). Naïve/memory score and effector score in PB CD8 + T cells of patients with AA and HDs. (B). Naïve/memory score in PB or BM CD8 + T cells of patients with AA and HDs was analyzed using GSE181989 and GSE145668. Naïve/memory scores in PB or BM CD8 + T cells of patients with AA were also tracked during the IST treatment (E-MTAB-9969). (C). The branched trajectory of CD8 + T cells grouped by individuals. (D). Expression maps showing log-normalized expression of marker genes ( IL7R , CCR7 , TCF7 , GZMK , GZMB , FCGR3A , PRF1 , and KLRD1 ) in the differentiation of Naïve CD8 + T into effector cells. Data are shown as log-normalized expressions. (E). The dot plot showing the expression levels of CCR4, CCR6, KLRB1 , and IL7R in CD8 + T cells from patients with AA and HDs. (F). The dot plot showing the expression levels of CCR4, CCR6, KLRB1 , and IL7R in CD8 + T cells from patients with AA and HD (GSE181989). (G). Representative flow cytometry depicting CD8 + cytotoxic cells expressing CCR4 and CCR6 in PB samples of patients with AA and HDs. (H). Representative flow cytometry density plots illustrating CD8 + T cells expressing CD127 in PB (i) and BM (ii) samples of AA patients and HDs.

Article Snippet: The nonspecific binding of the immunoglobulin to the Fc receptors of PBMCs and BMMCs was blocked by FcR Blocking Reagent (No. 130–0590901, Miltenyi Biotec), and stained with the following antibodies: FITC anti-human CD8a antibody (RPA-T8, No. 301050, BioLegend), APC-Cy7 anti-human CD4 antibody (A161A1, No. 357415, BioLegend), PE anti-human CCR4 (CD194) antibody (L291H4, No. 359412, BioLegend), APC anti-human CCR6 (CD196) antibody (G034E3, No. 353416, BioLegend), Biotin anti-human CD161 antibody (HP-3G10, No. 339932, BioLegend), APC anti-human CD30 antibody (BY88, No. 333910, BioLegend), Percp-Cy5.5 anti-human CD25 antibody (BC96, No. 302626, BioLegend), and PE-Cy7 anti-human CD127 antibody (A7R34, No. 25-1271-82, eBioscience), combined with APC-Cy7 Streptavidin (No. 405208, BioLegend), and PE/Cyanine7 Streptavidin (No. 405206, BioLegend).

Techniques: Expressing, Marker, Flow Cytometry

Journal: Heliyon

Article Title: Dysregulated T-cell homeostasis and decreased CD30 + Treg proliferating in aplastic anemia

doi: 10.1016/j.heliyon.2024.e35775

Figure Lengend Snippet: Impairment of Treg suppressive capacity and reduction in the proportion of CD30 + Treg cells in AA patients. (A). The violin plots show the expression levels of TNFSF8 in T cells of patients with AA and HD. (B). The dot plots depict the expression levels of TNFSF8 in PB CD8 + T and CD4 + T cells from patients with AA and HDs. (C) . ELISA was used to compare the serum levels of TNFSF8 in PB (AA, n = 22; HD, n = 11) and BM (AA, n = 15; HD, n = 8) samples from AA patients and HDs. (D ) The mRNA expression levels of TNFSF8 in granulocytes from the AABM (n = 4), AAPB (n = 5), HDPB (n = 5), and HDBM (n = 3) groups were assessed using quantitative polymerase chain reaction (qPCR) analysis, with statistical significance indicated as *P < 0.05, **P < 0.01, ***P < 0.001. (E). The proportion of CD30 + subset within various CD4 + T subsets (I: CD25low, II: CD127+CD25 + , III: CD25 + CD127-, IV:CD127-CD25high) enriched by CD25-beads from PB of a healthy donor was examined. (F). Representative flow cytometry dot plots were used to illustrate the presence of CCR4 and CD30 in CD4 + CD127-/lowCD25+ Treg cells in PB and BM samples from patients with AA and HDs.

Article Snippet: The nonspecific binding of the immunoglobulin to the Fc receptors of PBMCs and BMMCs was blocked by FcR Blocking Reagent (No. 130–0590901, Miltenyi Biotec), and stained with the following antibodies: FITC anti-human CD8a antibody (RPA-T8, No. 301050, BioLegend), APC-Cy7 anti-human CD4 antibody (A161A1, No. 357415, BioLegend), PE anti-human CCR4 (CD194) antibody (L291H4, No. 359412, BioLegend), APC anti-human CCR6 (CD196) antibody (G034E3, No. 353416, BioLegend), Biotin anti-human CD161 antibody (HP-3G10, No. 339932, BioLegend), APC anti-human CD30 antibody (BY88, No. 333910, BioLegend), Percp-Cy5.5 anti-human CD25 antibody (BC96, No. 302626, BioLegend), and PE-Cy7 anti-human CD127 antibody (A7R34, No. 25-1271-82, eBioscience), combined with APC-Cy7 Streptavidin (No. 405208, BioLegend), and PE/Cyanine7 Streptavidin (No. 405206, BioLegend).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: iScience

Article Title: Fat-to-blood recirculation of partially dysfunctional PD-1 + CD4 Tconv cells is associated with dysglycemia in human obesity

doi: 10.1016/j.isci.2024.109032

Figure Lengend Snippet:

Article Snippet: PE-Cy7 anti-human CD127 , Beckman Coulter , Cat#A64618; RRID: AB_2833031.

Techniques: Staining, Sequencing, Expressing, Software, Flow Cytometry, Cytometry

Journal: Cell

Article Title: Imprinted SARS-CoV-2-specific memory lymphocytes define hybrid immunity

doi: 10.1016/j.cell.2022.03.018

Figure Lengend Snippet:

Article Snippet: Anti-human CD127-PE/Cy7, Clone hIL7Rm21 , Becton Dickinson , CAT#560822; RRID:AB_2033938.

Techniques: Virus, Clinical Proteomics, Recombinant, Magnetic Beads, Luciferase, Cell Culture, Membrane, Plasmid Preparation, 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