NK cells were treated with 50 nM CMA or DMSO for three hours at 37°C. Cells were lysed as previously described (Fasbender et al., 2017 (link)) and analyzed by Western blotting using the following antibodies: anti-human perforin (Pf-344; MabTech), β-actin (4967; Cell Signaling), and anti-CD107a (H4A3; BD Biosciences).
Anti cd107a h4a3
Manufactured by BD
Anti-CD107a (H4A3) is a mouse monoclonal antibody that targets the CD107a (LAMP-1) surface antigen. CD107a is a marker of degranulation and can be used to identify activated cytotoxic T cells and natural killer cells. The antibody can be used in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd28 monoclonal antibody, by BD (2 mentions)
Golgistop, by BD (2 mentions)
Pf 344, by Mabtech (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Anti cd3 ucht1, by BioLegend (1 mentions)
Annexin v 7aad, by BioLegend (1 mentions)
Anti cd16 3g8, by BioLegend (1 mentions)
Lsrfortessatm flow cytometer, by BD (1 mentions)
Anti nkp44 p44 8, by BD (1 mentions)
Anti ifn γ 485 b3, by BioLegend (1 mentions)
Anti tnf α mab11, by Thermo Fisher Scientific (1 mentions)
Anti cd56 n901, by Beckman Coulter (1 mentions)
Anti nkp30 p30 15, by BioLegend (1 mentions)
Fix perm buffer set, by BioLegend (1 mentions)
Anti nkp46 29a1, by BioLegend (1 mentions)
Anti ki 67 16a8, by BioLegend (1 mentions)
Anti cd69 fn50, by BD (1 mentions)
Anti cd8 rpa t8, by BD (1 mentions)
Anti pd l1 mih1, by BD (1 mentions)
Fixable live dead stain, by Thermo Fisher Scientific (1 mentions)
11 protocols using anti cd107a h4a3
1
NK Cell Perforin Expression Analysis
2
Multiparametric Flow Cytometry Analysis
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Cell surface marker expression was analyzed on a Becton Dickinson LSR FortessaTM flow cytometer after staining with fluorochrome-conjugated antibodies. Intracellular staining was performed using the fix/perm buffer set (Biolegend), according to the manufacturer's instructions. Antibodies used were conjugated anti-NKp30 (P30–15, Biolegend), anti-NKp44 (P44–8.1, BD), anti-NKp46 (29A1.4, Biolegend), anti-IFN-γ (485.B3, Biolegend), anti-TNF-α (MAb11, eBioscience), anti-RANKL (MIH24, Biolegend), anti-Ki-67 (16A8, Biolegend), anti-CD107a (H4A3, BD), anti-CD57 (HCD57, Biolegend), anti-CD56 (N901, Beckman Coulter), anti-CD14 (HCD14, Biolegend), anti-CD16 (3G8, Biolegend), and anti-CD3 (UCHT1, Biolegend). Annexin V/7-AAD staining was performed according to the manufacturer's protocol (Biolegend). The data were analyzed using FlowJo v10.2 software.
3
Multiparametric Immune Profiling
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Fixable Live/Dead stains (Invitrogen) were utilized according to manufacturer’s instructions prior to surface staining. Following quenching and washing, surface staining was performed at 4 °C in RPMI media +1% FBS unless otherwise indicated. Antibody clones (manufacturer) utilized in this study: anti-CD8: RPA-T8 (BD), anti-CD107a: H4A3 (BD), anti-PD-1: EH12.2H7 (eBioscience), anti-PD-L1: MIH1 (BD), anti-CD69: FN50 (BD), anti-β2M: TU99 (BD), anti-CD19: HIB19 (BD). Data were collected on an LSR II (BD) and analyzed using FlowJo.
4
Expansion and Functional Analysis of SARS-CoV-2-specific T cells
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Approximately 1.5 × 106 PBMCs were stimulated with A*01/S865, A*02/S269 or A*03/S378-specific peptides (5 μM) and anti-CD28 monoclonal antibody (0.5 μg ml−1, BD) and expanded for 14 days in complete RPMI culture medium containing rIL-2 (20 IU ml−1, StemCell Technologies). Intracellular cytokine production and degranulation was assessed with spike-specific peptides (15 μM) in the presence of anti-CD107a (H4A3, 1:100) (BD Bioscience) for 1 h at 37 °C. Afterwards, brefeldin A (GolgiPlug, 0.5 μl ml−1) and monensin (GolgiStop, 0.5 μl ml−1) (all BD Biosciences) were added for additional 5 h, followed by surface and intracellular staining. The expansion capacity was calculated based on peptide-loaded HLA class I tetramer staining as previously described24 (link).
5
Antibody Protocols for Immunodetection
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For Western blotting, rabbit polyclonal anti-VAMP8 (V7389; Sigma-Aldrich), anti-phospho-p42/44 MAPK (Erk1/2, Thr202/Tyr204; 9101; Cell Signaling Technology), and anti-GAPDH (Cell Signaling Technology) antibodies were used. Secondary antibodies were HRP-conjugated donkey anti–rabbit (Invitrogen). For confocal microscopy, mouse monoclonal anti-perforin (δG9; BioLegend) and anti–granzyme B (GB11; BioLegend) as well as rabbit polyclonal anti-VAMP8 (W. Hong) antibodies were used. The VAMP8 antibody was generated using residues 1–75 of VAMP8, expressed as a fusion protein to GST (GST-endobrevin) and used to raise polyclonal antibodies against endobrevin. For TIRF microscopy and flow cytometric assays, mouse anti–human monoclonal anti-CD3 (BB11; Euroclone), mouse anti–human CD28 (CD28.2; BD), and mouse anti–human isotype IgG1 (MOPC-21; BD) antibodies were used for coating coverslips and stimulating cells. For flow cytometry stainings, fluorochrome-conjugated mouse anti–human monoclonal antibodies were used as follows: anti-CD3 (S4.1; Invitrogen), anti-CD4 (S3.5; Invitrogen), anti-CD8 (3B5; Invitrogen), anti-CD107a (H4A3; BD), and anti-FLAG (M2; GenScript).
6
Multiparametric Flow Cytometry Analysis
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Cells were acquired on an eight-colour FACSCanto II (BD Biosciences, Oxford, UK) and analysed with FlowJo (TreeStar, Ashland, OR, USA). Single cells of interest were gated based on their appearance in side and forward scatter area/height, exclusion of live/dead staining (fixable Aqua; Invitrogen) and surface staining. The following monoclonal antibodies (mAbs) were used for surface labelling: anti-CD3 (UCHT1), CD8 (HIT8a and SK1), CD16 (3G8), CD24 (ML5), CD44 (G44-26), GD2 (14.G2a) from BD Biosciences; anti-TCR-Vγ9 (Immu360) from Beckman Coulter, High Wycombe, UK; and anti-HLA-ABC (w6/32) from Biolegend, London, UK; together with appropriate isotype controls. Intracellular cytokines were detected using anti-IFN-γ mAbs (B27, BD Biosciences). Surface mobilisation of CD107a was detected by adding anti-CD107a (H4A3; BD Biosciences) mAbs and GolgiStop (BD Biosciences) to cultures for 5 h prior to flow cytometric analysis.
7
Cytokine Production Assay for NK Cells
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PBMCs were thawed and rested overnight in RPMI supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The PBMCs were co-cultured with K562 cells or anti-CD16-coated P815 cells for 12 h at an effector to target ratio of 10:1 in the presence of anti-CD107a (H4A3, BD Biosciences, San Jose, CA). Brefeldin A and monensin were added 1 h after co-incubation. For the anti-CD16 coating, P815 cells were incubated at 37°C with 10 μg/mL anti-CD16 antibody for 30 min. Cytokine production was detected by intracellular staining using antibodies to IFN-γ (B27) and TNF-α (Mab11) (all from BD Biosciences, San Jose, CA).
8
Expansion and Characterization of SARS-CoV-2-Specific T Cells
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After stimulation of approximately 1.5×106 PBMCs with A∗02/S269 (YLQPRTFLL) or A∗03/S378 (KCYGVSPTK) peptides (5 μM) and an anti-CD28 monoclonal antibody (0.5 μg ml-1, BD), the cells were expanded for 14 days in complete RPMI culture medium containing recombinant IL-2 (20 IU ml−1, StemCell Technologies). Intracellular cytokine production and degranulation was assessed with spike-derived peptides (15 μM) in the presence of anti-CD107a (H4A3, 1:100, BD Bioscience) for 1 h at 37 °C. Brefeldin A (GolgiPlug, 0.5 μl ml-1) and monensin (GolgiStop, 0.5 μl ml-1) (both BD Biosciences) were then added for an additional 4 h, followed by surface and intracellular staining. Calculation of the expansion capacity was based on peptide-loaded HLA class I tetramer staining as previously described.17
9
Activation and Identification of NK Cells
10
Cytotoxic T-cell Degranulation Assay
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The Peptivators CMV IE1 and pp65 (Milteniy Biotec, Bergisch Gladbach, Allemagne) were used to stimulate 6 h T cell degranulation following manufacture’s recommandations. Enriched KIR2DL3+ HLA-A2-pp65 specific T lymphocytes were pre-incubated with anti-CD107a (H4A3, BD Biosciences). T-cell degranulation was assessed after incubation for 5 h alone (negative control), or with different target cells (E:T ratio = 10:1) with brefeldin A (Sigma-Aldrich, Saint-Louis, MO, USA) at 10 μg/ml for the last 4 h. Cell surface staining was performed using the anti-human KIR 1F12 and GL183 mAbs and HLA-2-pp65 specific pentamer. To evaluate apoptosis in T lymphocytes, cells were incubated 3 h at 37 °C with target cells using a ratio 10:1 (E:T) before the staining with 7-AAD and Annexin-V (BD Biosciences). All flow cytometry data were collected using a FACSCalibur (BD Biosciences) and analyzed with Flowjo™ 10.2 software (LLC, Ashland, OR, USA).
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