Human PBMC were isolated from fresh buffy coat collected from healthy donors using the density gradient centrifugation method. Briefly, buffy coat was diluted with 2X volume PBS and was gently layered on top of an equal volume Ficoll-Paque PLUS (#17144003, Cytiva, Marlborough, MA), followed by centrifugation at 400 x g for 40 min without break. The PBMC were removed and transferred to a new 50 ml tube, washed in 25 ml DPBS buffer twice, and recovered by centrifugation at 350 x g for 10 min. The cells were counted and cultured in the presence of 1 µg/ml anti-human CD3 antibody (#317302, BioLegend, San Diego, CA) and 2 µg/ml anti-human CD28 antibody (#302902, BioLegend) for 72 h. The cells were then infected with NL4-3 at a MOI as indicated in the presence of 8 µg/ml polybrene by spinoculation at 850 x g, room temperature for 2 h. The cells were recovered by centrifugation, washed with fresh media, and continued to culture in the presence of 100 IU/ml human IL-2 (#21-8029-U050, Tonbo Biosciences, San Diego, CA) and used in subsequent Metformin experiments.
Human il 2
Manufactured by Cytek Biosciences
Human IL-2 is a cytokine that plays a crucial role in the activation and proliferation of T cells. It is an important component in various immunological processes and is commonly used in flow cytometry and other cell-based assays.
Automatically generated - may contain errors
2 protocols using human il 2
1
PBMC Isolation and T Cell Activation
2
Characterizing SIV-infected Rhesus Macaque T Cells
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Cells, previously isolated from rhesus macaque MLNs and cryopreserved, were infected with SIVmac239 and cultured in RPMI 1640 supplemented with 10% FBS and 40 U/mL of human IL-2 (Tonbo Biosciences, #21-8029). Uninfected cells were also cultured as the control group. Cultures were monitored for p27 expression using a commercial ELISA kit (Sino Biological, KIT11695, Beijing, China). After infection was confirmed by a positive ELISA test, cells were harvested and blocked with a solution of PBS-T containing 3% BSA and human Fc block (BD #564219) for 30 min on ice. Cells were then washed and successively stained with 1 or 10 μg/mL AvFc and goat anti-human IgG-FITC secondary antibodies (Abcam #ab97224). Flow cytometry was performed on the BD FACScan system. Captured cell populations were gated for live lymphocytes, and unstained cells were used to determine the background fluorescence.
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