After washing the PBMCs in FACS buffer (phosphate-buffered saline, 1% fetal bovine serum, and 0.5 mmol/l ethylenediaminetetraacetic acid), the following antibodies were used for flow cytometry: VioBlue-conjugated anti-human CD4 (clone VIT4; Miltenyi Biotec, Bergisch Gladbach, Germany), FITC-conjugated anti-human CD8 (clone RPA-T8; BD Biosciences, Heidelberg, Germany) and CD25 (clone B1.49.9; Beckman Coulter, Marseille, France), APC-conjugated anti-human PD-1 (clone EH12.2H7; Biolegend, San Diego, CA) and CD45RA (clone HI100; Biolegend), and PE-conjugated anti-human Tim-3 (clone F38-2E2; Biolegend). After staining, the cells were washed in FACS buffer and analyzed using a MACSQuant flow cytometer with MACSQuantify software (Miltenyi Biotec). In this study, the percentages of PD-1+ and Tim-3+ T cells were calculated as percentages of the total CD4+ or CD8+ T cells. Tregs were identified as CD4+ CD45RA- CD25high cells [26 (link)] and were calculated as a percentage of the CD4+ lymphocytes. MDSCs were identified as CD11b + CD33+ cells [27 (link)] and were calculated as a percentage of the total PBMCs.
Cd45ra clone hi100
Manufactured by BioLegend
CD45RA (clone HI100) is a mouse monoclonal antibody that recognizes the CD45RA isoform of the CD45 protein, which is expressed on the surface of certain types of leukocytes, including naive T cells and memory B cells. This antibody is commonly used in flow cytometry applications to identify and quantify these cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Macsquant flow cytometer, by Miltenyi Biotec (1 mentions)
Macsquantify software, by Miltenyi Biotec (1 mentions)
Cd8 clone rpa t8, by BioLegend (1 mentions)
Ccr7 clone g043h7, by BioLegend (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Facscanto 2 device, by BD (1 mentions)
Ifn γ clone 4s b3, by BioLegend (1 mentions)
Cd8a clone rpa t8, by BioLegend (1 mentions)
Clone tw4 2f8, by BioLegend (1 mentions)
Facsdiva software, by BD (1 mentions)
Mouse foxp3 buffer set, by BD (1 mentions)
3 protocols using cd45ra clone hi100
1
Multiparametric Flow Cytometry Analysis
2
Phenotypic Analysis of Expanded T Cells
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T cells selected and expanded in vitro were characterized using the following fluorochrome-conjugated monoclonal antibodies: APC-conjugated CD3 (clone UCHT1; BioLegend) and CD8+ (clone RPA-T8; BioLegend); FITC-conjugated CD16 (clone 3G8; BioLegend) and CCR7 (clone G043H7; BioLegend); PerCP-Cy5.5-conjugated CD56 (clone B159; BD Biosciences) and CD45RA (clone HI100; BioLegend); BV421-conjugated CD8+ (clone RPA-T8; BD Biosciences); APC-H7-conjugated CD4+ (clone RPA-T4; BD Biosciences); PE-conjugated CD4+ (clone RPA-T4; BioLegend). For the tetramer staining, CTLs were incubated with the PE-conjugated HLA-A*0201-DEPDC1#5 or PE-conjugated HLA-A*0201-Melan-A26–35*A27L tetramers. Samples were analyzed by an LSRII flow cytometer (BD Biosciences) and evaluated with FlowJo software (TreeStar, Inc.).
3
Multicolor Flow Cytometry for Mouse and Human Immune Markers
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Directly conjugated mAb for the following mouse antigens were purchased from BD Bioscience if not stated otherwise: CD3 (clone 145-2C11), CD4 (clone GK1.5), CD8 (clone 53-6.7), CD11a, (clone 2D7), CD11b (clone M1/70), CD11c (clone HL3), CD40L (clone MRI; Miltenyi Biotec, Bergisch Gladbach, Germany), CD69 (clone H1.2F3), and Foxp3 (clone MF23). Before intranuclear staining of forkhead box protein (FoxP)3, cells were fixed and permeabilized using the Mouse Foxp3 Buffer Set (BD Bioscience) according to the manufacturer’s instructions.
Directly conjugated mAb for the following human antigens was purchased from BioLegend (San Diego, CA): C-C chemokine receptor (CCR)7 (clone G043H7), CD4 (clone RPA-T4), CD45RA (clone HI100), CD8a (clone RPA-T8), Foxp3 (clone 259D), IFNγ (clone 4S.B3), IL-10 (clone JES3-9D7), latency-associated protein addressing TGFβ (clone TW4-2F8), and TNFa (clone Mab11). Before intracellular cytokine or FoxP3 staining, cells were fixed and permeabilized using the FIX & PERM® Cell Fixation & Cell Permeabilization Kit (BioLegend) according to the manufacturer’s instructions. Stained cells were assessed by multicolor flow cytometry using a FACS Canto II device with the FACS Diva software (both from BD Biosciences). During data analysis by the FlowJo software (FlowJo, LLC, Ashland, Oregon), viable lymphocytes were defined by their forward/sideward scatter properties.
Directly conjugated mAb for the following human antigens was purchased from BioLegend (San Diego, CA): C-C chemokine receptor (CCR)7 (clone G043H7), CD4 (clone RPA-T4), CD45RA (clone HI100), CD8a (clone RPA-T8), Foxp3 (clone 259D), IFNγ (clone 4S.B3), IL-10 (clone JES3-9D7), latency-associated protein addressing TGFβ (clone TW4-2F8), and TNFa (clone Mab11). Before intracellular cytokine or FoxP3 staining, cells were fixed and permeabilized using the FIX & PERM® Cell Fixation & Cell Permeabilization Kit (BioLegend) according to the manufacturer’s instructions. Stained cells were assessed by multicolor flow cytometry using a FACS Canto II device with the FACS Diva software (both from BD Biosciences). During data analysis by the FlowJo software (FlowJo, LLC, Ashland, Oregon), viable lymphocytes were defined by their forward/sideward scatter properties.
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