T cells were labeled with 1 M CFSE (Invitrogen, Grand Island, NY), cultured with CD3/CD28 beads (Invitrogen, Grand Island, NY) for 3 days, stained with antibodies against CD4 or CD8 (Beckman Coulter, Indianapolis, IN) and fixed for flow cytometric analysis on a FACS Calibur (BD Biosciences). Percent proliferation was determined based on dilution of CFSE in CD4+ or CD8+ cells. For analysis of T cell activation checkpoint receptor expression, T cells were stained with CD4-APC, CD8-FITC (Beckman Coulter) and PD-1 or CTLA-4 antibodies (BD Biosciences, San Jose, CA) after 3 days of culture with CD3/CD28 beads. For analysis of apoptosis, cells were stained with APC-Annexin V (BD Biosciences, San Jose, CA) and propidium iodide (BD Biosciences, San Jose, CA) as recommended by the manufacturer.
Cd3 cd28 beads
Manufactured by BD
CD3/CD28 beads are a type of lab equipment used for cell activation and expansion. They contain antibodies for the CD3 and CD28 cell surface proteins, which are important for T cell stimulation and proliferation. The beads provide a controlled and consistent way to activate T cells in vitro.
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Lab products found in correlation
2 protocols using cd3 cd28 beads
1
T Cell Proliferation and Checkpoint Receptor Analysis
2
Assessing NK Cell Cytotoxicity and Degranulation
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NK cell degranulation was performed as described (26 (link)). Briefly: Freshly isolated PBMCs were stimulated with either with medium alone or K562 cells (lacking MHC 1 expression) for 2.5 h in presence of anti-CD107a-PE (BD Biosciences, Heidelberg, Germany). Lytic exocytosis of NK cells (CD3- CD56+) are measured by CD107a (CD107a–PE (H4A3, IgG1) degranulation.
Cytotoxicity was measured by stimulating freshly isolated PBMCs by standard Chromium-51 release assay. K562 target cells were labeled with 51Cr and incubated with PBMCs. Supernatant was harvested after 4 h, transferred to lumaplates and dried overnight. Radioactivity was determined with TopCount NXT. NK cell percentage was measured by FACS staining and NK to target ratio was calculated. T cell proliferation was measured by CFSE dilution after 5–7 days of stimulation with medium alone or PHA (1.25 μg/ml) or CD3 (300 ng/ml) with CD28 (1,000 ng/ml) or CD3/CD28 Beads (BD Biosciences) as previously described (27 (link)).
Cytotoxicity was measured by stimulating freshly isolated PBMCs by standard Chromium-51 release assay. K562 target cells were labeled with 51Cr and incubated with PBMCs. Supernatant was harvested after 4 h, transferred to lumaplates and dried overnight. Radioactivity was determined with TopCount NXT. NK cell percentage was measured by FACS staining and NK to target ratio was calculated. T cell proliferation was measured by CFSE dilution after 5–7 days of stimulation with medium alone or PHA (1.25 μg/ml) or CD3 (300 ng/ml) with CD28 (1,000 ng/ml) or CD3/CD28 Beads (BD Biosciences) as previously described (27 (link)).
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