Degranulation assay of NK cells following GAD65 AA 114–122 peptide presentation was performed through quantification of cell-surface CD107a expression by FACS analysis [40 (link)]. Briefly, at the end of the co-culture period, culture plate was centrifuged at 2000 rpm for 2 minutes and cell staining was directly performed by adding the mixture of mouse mAbs anti- human CD3 Alexa Fluor 700-A (1:50, BD), CD56 PECy7 (1:50, BD), CD8 PECy5 (1:30, clone RPA-T8, cat# 557746, BD), ILT2 (APC,1:50, eBioscience) and CD107a FITC (1:10, clone H4A3, cat# 555800, BD).
Clone h4a3
Manufactured by BD
The Clone H4A3 is a laboratory equipment product designed for cell culture applications. It functions as a tool for the isolation, cultivation, and maintenance of cells. The product specifications and technical details are available upon request.
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Lab products found in correlation
Golgistop, by BD (2 mentions)
Cd56 pe cy7, by BD (1 mentions)
Cd8 pe cy5, by BD (1 mentions)
Rpa t8, by BD (1 mentions)
Cd107a fitc, by BD (1 mentions)
Golgiplug, by BD (1 mentions)
Hril 15, by R&D Systems (1 mentions)
Clone rpa t8, by BD (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Anti cd56 pe, by Beckman Coulter (1 mentions)
Fitc conjugated isotype control, by BD (1 mentions)
Mouse igg2b, by R&D Systems (1 mentions)
Accuri cflow, by BD (1 mentions)
Mab1300, by R&D Systems (1 mentions)
Mab1599, by R&D Systems (1 mentions)
4 protocols using clone h4a3
1
NK Cell Degranulation Assay with GAD65 Peptide
2
Quantifying T Cell Degranulation
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Cell-surface expression of CD107a was used as a marker for T cell degranulation (38 (link)). Redirected cytotoxicity assays were performed as described above, with the following modification [as previously described in (18 (link))]. After plating the target cells, effector cells, and DART molecules, the plate was incubated for 18 h and then FITC- or APC-Cy7-CD107a antibody (clone H4A3, BD Biosciences), brefeldin A (GolgiPlug, 1 μL/mL, BD Biosciences), and monensin (GolgiStop, 4 μL/6mL, BD Biosciences) were added to each well and the plates were incubated for an additional 6 h. Plates were then washed and stained with a viability marker and fluorescently conjugated antibodies as described above (phenotypic characterization of T cells), or with a truncated staining panel that included only antibodies specific for CD3, CD4, and CD8, using standard techniques. Data analyses were performed using FlowJo software (v10.5.3).
3
Cytotoxicity and Cytokine Assay of Activated CD8+ T Cells
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Frozen PBMCs were thawed and cultured at 2 × 106 cells/mL overnight in RPMI supplemented with 5% autologous plasma, 40 U/mL of human recombinant (hr) interleukin (IL)-2 (Roche), 20 ng/mL of hrIL-7 (R&D Systems), and 10 ng/mL of hrIL-15 (R&D Systems) to obtain armed-CD8+ T lymphocytes. Then, CD3+ T cells were purified by negative selection (CD3+CD56+ NKT Cell Isolation Kit, human; Miltenyi Biotech), and 4 × 105 CD3+ lymphocytes were stained with APC anti-CD107a/LAMP-1 (BD Biosciences, clone H4A3) and stimulated in the presence or not of anti-CD3/CD28 (1 bead/cell) for 4 hours. Brefeldin A (Sigma-Aldrich) was added at 10 μg/mL for the last 3 hours of cell incubation. Then, CD3+ T cells were stained with a combination of the following fluorochrome-conjugated antibodies FITC anti-human CD8 (BD Biosciences, Clone RPAT8), V500 anti-human CD45RA (BD Biosciences, Clone HI100), BB700 anti-human CCR7, PE anti-human IFN-γ (BD Biosciences, Clone B27-RUO), and BV421 anti-human Granzyme B to measure cytotoxicity and cytokine production. Intracytoplasmic molecules were evaluated by using the fixation/permeabilization solution kit BD Cytofix-Cytoperm (BD Biosciences), according to the manufacturer's instructions. For the evaluation of CD107a/LAMP-1 and IFN-γ positive events, the medium sample was used for setting the gate. The gating strategy used is shown in eFigure 2.
4
NK Cell Degranulation Assay: Comprehensive Protocol
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NK degranulation assays were performed in a similar manner to that described previously [39] , [81] (link). Briefly, PBMC (approved by the Cardiff University School of Medicine Ethics Committee Ref. no: 10/20) and incubated overnight with IFN-α (1000 IU/ml) and IL-15 (15 ng/ml, Milenyi Biotech). PBMC (0.5–1×106) were incubated for 6 h with 0.5–1×105 fibroblast targets per well in a 96 well plate at an effector∶target (E∶T) ratio of 10∶1, with the addition of 3 µl per well FITC-conjugated anti-CD107 antibody (cat. no. 555800, clone H4A3, BD Biosciences) or 3 µl per well FITC-conjugated isotype control (cat. no. 555748, BD Biosciences), adding 1 µl/well BD GolgiStop (BD Biosciences) 1 h after beginning the incubation. (For antibody blocking experiments, targets were pre-incubated with anti-MICA (Clone 159277, mouse IgG2B, MAB1300, R&D Systems) or isotype control (MICB non-blocking antibody, Clone 236511, mouse IgG2B, MAB1599, R&D Systems) antibodies at a concentration of 10 µg/ml for 30 min prior to incubation with PBMC). PBMC were harvested and stained with conjugated antibodies against CD3 (anti-CD3 PE-Cy7, cat. no. 737657, Beckman Coulter, High Wycombe, UK) and CD56 (anti-CD56 PE, cat. no. A07788, Beckman Coulter), and fixed in 2% PFA before analysis by flow cytometry (BD Biosciences Accuri C Flow) (Fig. S10A ).
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