Ex vivo (d0) and peptide-expanded (d6) CD8+ T cells were isolated from PBMCs and LNMCs using EasySep Human CD8+ T Cell Isolation Kit (StemCell Technologies) and stimulated with 4 nM APC-pHLA tetramer at 37°C for 4 hours in R10 with BV711-anti-CD107A (clone H4A3, Biolegend) to measure cytolytic degranulation. Cells were stained with BUV395-anti-CD8 (clone RPA-T8, BD Biosciences), Live/Dead Blue (Thermo Fisher), fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences), and stained for intracellular PE-anti-perforin (clone B-D48, Biolegend) and PE-CF594-anti-granzyme B (clone GB11, BD Biosciences). FMO controls were used to establish gating. Experimental negative controls were pre-treated with 50 nM dasatinib 30 minutes before tetramer stimulation to prevent activation, and experimental positive controls were treated with PMA and ionomycin (eBioscience Cell Stimulation Cocktail, Thermo Fisher). Samples were analyzed by flow cytometry. Representative gating and controls are shown in fig. S5A and C . Samples with fewer than 40 live HIV-specific pHLA-tetramer+ CD8+ T cells at d0 in both PB and LN were excluded from downstream analyses.
Bv711 anti cd107a clone h4a3
Manufactured by BioLegend
BV711-anti-CD107A (clone H4A3) is a fluorochrome-conjugated antibody that recognizes the cell surface protein CD107A (also known as LAMP-1). It can be used for the detection and analysis of CD107A-expressing cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Ebioscience cell stimulation cocktail, by Thermo Fisher Scientific (2 mentions)
Pe anti perforin clone b d48, by BioLegend (2 mentions)
Pe cf594 anti granzyme b clone gb11, by BD (2 mentions)
Buv395 anti cd8 clone rpa t8, by BD (2 mentions)
Cytofix cytoperm, by BD (2 mentions)
Live dead blue, by Thermo Fisher Scientific (2 mentions)
Easysep human cd8 t cell isolation kit, by STEMCELL (2 mentions)
2 protocols using bv711 anti cd107a clone h4a3
1
Isolation and Phenotyping of CD8+ T Cells
2
Isolation and Phenotyping of CD8+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Ex vivo (d0) and peptide-expanded (d6) CD8+ T cells were isolated from PBMCs and LNMCs using EasySep Human CD8+ T Cell Isolation Kit (StemCell Technologies) and stimulated with 4 nM APC-pHLA tetramer at 37°C for 4 hours in R10 with BV711-anti-CD107A (clone H4A3, Biolegend) to measure cytolytic degranulation. Cells were stained with BUV395-anti-CD8 (clone RPA-T8, BD Biosciences), Live/Dead Blue (Thermo Fisher), fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences), and stained for intracellular PE-anti-perforin (clone B-D48, Biolegend) and PE-CF594-anti-granzyme B (clone GB11, BD Biosciences). FMO controls were used to establish gating. Experimental negative controls were pre-treated with 50 nM dasatinib 30 minutes before tetramer stimulation to prevent activation, and experimental positive controls were treated with PMA and ionomycin (eBioscience Cell Stimulation Cocktail, Thermo Fisher). Samples were analyzed by flow cytometry. Representative gating and controls are shown in fig. S5A and C . Samples with fewer than 40 live HIV-specific pHLA-tetramer+ CD8+ T cells at d0 in both PB and LN were excluded from downstream analyses.
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