Lymphocyte proliferative response to mitogens and the amount of TCR excision circles were determined as previously described (Amariglio et al., 2010 (link)). Proliferative response was measured by labeling PBMCs with 2.5 mM CFSE (Thermo Fisher Scientific), 7-aminoactinomycin D (BD Biosciences), PacB anti-CD3 (SK7; Biolegend), PE anti-CD25 (M-A251; BD Biosciences), APC-H7 anti-CD8 (SK1; BD Biosciences), and BV711 anti-CD4 (SK3; BD Biosciences) 4 d after stimulation. Activation response was determined by CD25 levels in stimulated PBMCs. The analysis of TCR Vβ expression was determined according to manufacturer’s manual (Beta Mark TCR Vβ Repertoire Kit; Beckman Coulter). NK degranulation was assessed by CD107a surface staining without stimulation (medium-cultured cells) and 3 h after stimulation with K562 cells at a ratio of 1:1 as previously described (Bryceson et al., 2012 (link)). The erythroleukemic cell line K562 (CCL-243; ATCC) was used as a target cell line. NK cells were cultured in medium containing 600 U/ml IL-2 (Novartis) for 48 h to assess the degranulation of activated NK cells. For surface staining, the following antibodies were used: PacB anti-CD3 (SK7; Biolegend), APC-H7 anti-CD8 (SK1; BD Biosciences), FITC anti-CD56 (NCAM16.2; BD Biosciences), and PE anti-CD107a (H4A3; BD Biosciences).
Anti cd8 apc h7 sk1
Manufactured by BD
Sourced in United States
Anti-CD8 APC-H7 (SK1) is a fluorescently-labeled antibody that binds to the CD8 surface marker. It is designed for use in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Live dead aqua, by Thermo Fisher Scientific (2 mentions)
Anti cd26 fitc ba5b, by BioLegend (2 mentions)
Anti cd161 pe cy7 hp 3g10, by BioLegend (2 mentions)
Fix perm medium b, by Thermo Fisher Scientific (2 mentions)
Anti cd3 alexafluor700 ucht1, by BioLegend (2 mentions)
Anti ifn γ pe dazzle594 4s b3, by BioLegend (2 mentions)
Anti il 17 bv421, by BioLegend (2 mentions)
Fix perm medium a, by Thermo Fisher Scientific (2 mentions)
Anti cd4 bv711 okt4, by BioLegend (2 mentions)
Lsrfortessa, by BD (2 mentions)
Anti cd3 apc sk7, by BD (1 mentions)
Anti hla dr pe, by Beckman Coulter (1 mentions)
Anti cd27 percp cy 5, by BD (1 mentions)
Beta mark tcr vβ repertoire kit, by Beckman Coulter (1 mentions)
Pe anti cd107a h4a3, by BD (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by Novartis (1 mentions)
7 aminoactinomycin d, by BD (1 mentions)
Anti cd3 bv510, by BioLegend (1 mentions)
Anti cd3 percp, by BD (1 mentions)
5 protocols using anti cd8 apc h7 sk1
1
Lymphocyte Proliferation and NK Cell Degranulation
2
Phenotyping of MAIT Cells from BAL and PBMCs
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When sufficient cells were available, freshly isolated matched BAL lymphocytes and PBMCs were simultaneously stimulated using PMA (25 ng/mL) and ionomycin (500 ng/mL) for 6 hours at 37°C in 96-well microplates. After 6 hours, plates were washed in phosphate buffered saline (PBS) before cells were stained with 1:500 of either MR1 6-FP (PE) or MR1 5-OP-RU (PE) tetramer (NIH Tetramer Core Facility). Tetramer staining was performed in the dark at room temperature for 40 minutes, followed by surface staining at 4°C for 20 minutes. The surface staining antibody master-mix included: 1:25 of anti-CD4 BV711 (OKT4, BioLegend), 1:100 of anti-CD8 APC-H7 (SK1, BD Biosciences), 1:50 of anti-CD26 FITC (BA5b, BioLegend), 1:50 of anti-CD161 PE-Cy7 (HP-3G10, BioLegend) and 1:800 of a fixable viability dye (Live/Dead Aqua, Invitrogen). Cells were then fixed (Fix & Perm Medium A, Invitrogen) at room temperature in the dark for 15 minutes and permeabilized (Fix & Perm Medium B, Invitrogen) for 20 minutes at 4°C. Intracellular antibodies were added during permeabilization and included 1:50 each of: anti-CD3 AlexaFluor700 (UCHT1, BioLegend), anti-IFN-γ PE-Dazzle594 (4S.B3, BioLegend), anti-granzyme B AlexaFluor647 (GB11, Biolegend), and anti-IL-17 BV421 (BL168, BioLegend). Cells were PBS washed, acquired using the LSRFortessa™ (BD Biosciences) and the data then analyzed using FlowJo (v10.4).
3
T Lymphocyte Differentiation Profiling
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To define T lymphocytes differentiation status, both CTL and Th lymphocyte subsets were distinguished from CD3+CD8+ and CD3+CD4+ gate, respectively as follows: activated T lymphocytes (HLA-DR+), naïve (N; CD45RA+CD27+), central memory (CM; CD45RA−CD27+), effector memory (EM; CD45RA−CD27−) and effectors (E; CD45RA+CD27−) were evaluated using anti-CD45RA-FITC (L48, BD Bioscience, San Jose, CA, USA), anti-CD3-APC (SK7, BD Bioscience, San Jose, CA, USA), anti-CD8-APCH7 (SK1, BD Bioscience, San Jose, CA, USA), anti-CD4-V450 (RPA-T4, BD Bioscience, San Jose, CA, USA), anti-CD27-PerCP-Cy ™ 5.5 (L128, BD Bioscience, San Jose, CA, USA), and anti-HLA-DR-PE (L243, Beckman Coulter, Chaska, MN, USA). Gating strategies are shown in Supplementary Material
4
Multiparameter Analysis of T Cell Responses
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When sufficient cells were available, freshly isolated matched BAL lymphocytes and PBMCs were simultaneously stimulated using PMA (25 ng/mL) and ionomycin (500 ng/mL) for 6 hours at 37°C in 96-well microplates. After 6 hours, plates were washed in phosphate buffered saline (PBS) before cells were stained with 1:500 of either MR1 6-FP (PE) or MR1 5-OP-RU (PE) tetramer (NIH Tetramer Core Facility). Tetramer staining was performed in the dark at room temperature for 40 minutes, followed by surface staining at 4°C for 20 minutes. The surface staining antibody master-mix included: 1:25 of anti-CD4 BV711 (OKT4, BioLegend), 1:100 of anti-CD8 APC-H7 (SK1, BD Biosciences), 1:50 of anti-CD26 FITC (BA5b, BioLegend), 1:50 of anti-CD161 PE-Cy7 (HP-3G10, BioLegend) and 1:800 of a fixable viability dye (Live/Dead Aqua, Invitrogen). Cells were then fixed (Fix & Perm Medium A, Invitrogen) at room temperature in the dark for 15 minutes and permeabilised (Fix & Perm Medium B, Invitrogen) for 20 minutes at 4°C. Intracellular antibodies were added during permeabilization and included 1:50 each of: anti-CD3 AlexaFluor700 (UCHT1, BioLegend), anti-IFN-γ PE-Dazzle594 (4S.B3, BioLegend), anti-granzyme B AlexaFluor647 (GB11, Biolegend) and anti-IL-17 BV421 (BL168, BioLegend). Cells were PBS washed, acquired using the LSRFortessa™ (BD Biosciences) and the data then analysed using FlowJo (v10.4).
5
Comprehensive T-cell Subset Analysis
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Blood T-cell subsets from 23 patients were analyzed by flow cytometry. First, patient peripheral blood was lysed (lysing buffer, BD Biosciences), and Fc was blocked (BD Biosciences). Then, CD8 + T-cell and Treg subsets were identified with a panel of antibodies: BV-510-anti-CD3 (HIT3a), APC-H7-anti-CD4 (RPA-T4), BUV395-anti-CD8 (RPA-T8), BB515-anti-CD38 (HIT2), BV786-anti-CD45RO (UCHL1), PE-anti-CD137 (4B4-1) and AF-647-anti-PD-1 (NAT105, BioLegend). After extracellular staining, the cells were fixed and permeabilized with BB700-anti-FOXP3 (236A/E7), BV650-anti-IFN-γ (4S.B3) and PE-Cy7-anti-Ki-67 (B56) according to the manufacturer’s protocol. The stained cells were washed, resuspended and analyzed with an LSRFortessa flow cytometer (BD Biosciences). All antibodies used for flow cytometry analysis except anti-PD-1 were purchased from BD Biosciences.
To isolate CD8 + T cells and Tregs for RNA sequencing (RNA-seq), patient PBMCs were stained with PerCP-anti-CD3 (SK7), PE-Cy7-anti-CD4 (RPA-T4), APC-H7-anti-CD8 (SK1), BB515-anti-CD25 (2A3), AF-647-anti-CD127 (HIL-7R- M21) and 7-AAD (BD Pharmingen), and then, the resuspended cells were sorted with a FACSAria III flow cytometer. All antibodies used were purchased from BD Biosciences.
To isolate CD8 + T cells and Tregs for RNA sequencing (RNA-seq), patient PBMCs were stained with PerCP-anti-CD3 (SK7), PE-Cy7-anti-CD4 (RPA-T4), APC-H7-anti-CD8 (SK1), BB515-anti-CD25 (2A3), AF-647-anti-CD127 (HIL-7R- M21) and 7-AAD (BD Pharmingen), and then, the resuspended cells were sorted with a FACSAria III flow cytometer. All antibodies used were purchased from BD Biosciences.
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