PBMCs were resuspended in complete medium (RPMI 1640 supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin/streptomycin) at 1 × 107 cells/mL and cultured at 1 × 106 cells/well in 96-well V-bottom plates (Corning) with the relevant peptides (each at 0.5 μg/mL) for 30 min prior to the addition of anti-CD28/CD49d (3 μL/mL; clone L293/L25; BD Biosciences), brefeldin A (1 μL/mL; Sigma-Aldrich), monensin (0.7 μL/mL; BD Biosciences), and anti-CD107a–PE-CF594 (clone H4A3; BD Biosciences). Negative control wells lacked peptides, and positive control wells included staphylococcal enterotoxin B (SEB; 0.5 μg/mL; Sigma-Aldrich) or plate-bound anti-CD3 (1 μg/mL; clone OKT3; BioLegend). Cells were analyzed by flow cytometry after incubation for 8 hr at 37°C.
Anti cd107a pe cf594 clone h4a3
Manufactured by BD
Anti-CD107a–PE-CF594 (clone H4A3) is a fluorescently-labeled monoclonal antibody that binds to the CD107a (LAMP-1) cell surface antigen. CD107a is a marker of degranulation and can be used to identify activated immune cells.
Automatically generated - may contain errors
Lab products found in correlation
Brefeldin a, by Merck Group (2 mentions)
Monensin, by BD (2 mentions)
96 well 5 bottom plates, by Corning (1 mentions)
Clone okt3, by BioLegend (1 mentions)
Clone l293 l25, by BD (1 mentions)
Staphylococcal enterotoxin b, by Merck Group (1 mentions)
V bottom plate, by Corning (1 mentions)
Anti cd49d clone l25, by BD (1 mentions)
2 protocols using anti cd107a pe cf594 clone h4a3
1
Cytokine Production Assay of PBMCs
2
Stimulating and Analyzing PBMCs
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PBMCs were seeded in complete medium at 2 x 10 6 cells/mL in 96-well V-bottom plates (Corning) with unconjugated anti-CD28 (clone L293) and anti-CD49d (clone L25) (each at 3 µL/mL; BD Biosciences), brefeldin A (1 µL/mL; Sigma-Aldrich), monensin (0.7 µL/mL; BD Biosciences), anti-CD107a-PE-CF594 (clone H4A3; BD Biosciences), and the relevant viral peptides (each at a final concentration of 0.5 µg/mL). Unstimulated negative controls were included in each assay. Cells were analyzed by flow cytometry after incubation for 6 hours at 37°C.
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