Peripheral blood (PB) from de-identified healthy donors was collected in this study after obtaining informed consent. This study was approved by the IRB committee of Temple University. Naïve CD4+ T cells and CD8+ T cells were obtained from PB mononuclear cells (PBMCs) through negative selection using human naïve T cell isolation kit II from Miltenyi Biotec (San Diego, CA, USA). T cells were activated by anti-CD3 Ab + anti-CD28 Ab at day 0. D2HG – ranging from 0 to 500 μM concentrations – was added to the culture on day 0. Cells were collected at different time points of culture, counted, and analyzed for intracellular staining of IFN-γ using an intracellular staining kit from BD (BD Cytofix/Cytoperm™ Kit, BD Biosciences, CA, USA) and antibodies from Biolegend. Multi-color flow cytometric analysis was performed using a BD LSRII flow cytometer as described (25 (link)).
Human na ve t cell isolation kit 2
Manufactured by Miltenyi Biotec
Sourced in United States, Germany
The Human Naïve T Cell Isolation Kit II is a product designed for the isolation of naïve T cells from human peripheral blood mononuclear cells (PBMCs). The kit utilizes a magnetic-bead-based negative selection approach to separate naïve T cells from other cell types present in the PBMC population.
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2 protocols using human na ve t cell isolation kit 2
1
Modulation of T Cell Activation by D2HG
2
In vitro Generation of Regulatory T Cells
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For in vitro generation of Tregs, naïve T cells was isolated by human naïve T cell isolation kit II (Miltenyi, Koln, Germany), and further stimulated with human recombined human TGF-β (1ng/mL) (Peprotech, NJ ) for 7 days. Dead cells were excluded based on staining with Live/Dead fixable dye. After that, cells were, fixed, permeabilized with IntraPrep reagent (BD Pharmingen, CA, USA), and then stained with fluorochromeconjugated labeled antibodies including CD4 (561833), CD25 (557192), Foxp3 (560082) (BD Pharmingen, CA, USA). The Tregs were determined gated with CD4 and further defined as CD25 and Foxp3 positive cells. Data were acquired on BD FACSVerse™ flow cytometer (BD Pharmingen, CA, USA).
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