Peripheral blood mononuclear cells (PBMC) were isolated by standard Ficoll density gradient centrifugation. As described before50 (link), CD4+ T cells were purified from PBMC using magnetic negative selection (Miltenyi Biotech), cultured in Xvivo15 supplemented with 10% inactivated human AB serum, and activated in vitro with Phytohemagglutinin (PHA) (2.5 μg/ml; Sigma, St Louis, MO) and IL-2 (50 U/ml) (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Human rIL-2 from Dr. Maurice Gately, Hoffmann-La Roche Inc)51 (link) for 1 to 3 days prior to HIV-infection. Purity higher than 98% was obtained after CD4+ T cell magnetic isolation.
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Isolation and Activation of CD4+ T Cells for HIV Infection Study
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Corresponding Organization : Dartmouth College
Other organizations : University of Alabama at Birmingham
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Variable analysis
independent variables
- Phytohemagglutinin (PHA) (2.5 μg/ml)
- IL-2 (50 U/ml)
- HIV-infection
- Peripheral blood mononuclear cells (PBMC) isolated by Ficoll density gradient centrifugation
- CD4+ T cells purified from PBMC using magnetic negative selection
- CD4+ T cells cultured in Xvivo15 supplemented with 10% inactivated human AB serum
- Purity of CD4+ T cells higher than 98% after magnetic isolation
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