A3R5.7 cells were acquired from the National Institutes of Health (NIH) AIDS Reagent Program. A3R5.7 cells were derived from A3.01, which naturally expresses CD4, CXCR4, and α4β7. HIV-1(AD8) was provided by M. A. Martin. Virus stocks of NLENG1-ES-IRES(NL4-3), NLENG1-ES-IRES(Yu2), and HIV-1(AD8) were prepared by transfection of human embryonic kidney (HEK) 293 T cells with cloned proviral DNA as described (9, 49). Viral titer (TCID50) was determined on the Rev-dependent green fluorescent protein (GFP) indicator cell (51), Rev-A3R5-GFP (Virongy, Manassas, VA). For viral infection of resting CD4 T cells, cells were infected with envelope-negative GFP reporter HIV-1 virus NLENG1-ES-IRES, pseudo-typed with NL4-3 or YU2 envelope. Infection was performed by spinoculation for 2 hours at 1200g of 400 virions particles per cell at 37°C, in the presence of DEAE Dextran (5 μg/ml; Sigma-Aldrich). After infection, cells were washed and incubated for 6 days with or without IL-7 (25 ng/ml; R&D Systems). T cells were stained with anti–CD45RO-Pacific Blue monoclonal antibody (BD Biosciences, San Jose, CA) and analyzed by flow cytometry for CD45RO and GFP expression. For treatment of resting CD4 T cells with HIV(AD8), cells were pretreated with or without PTX (100 ng/ml; Sigma-Aldrich) for 1 hour at 37°C and then treated with HIV(AD8) (103.5 to 104.5 TCID50 per million cells) for various times. Cells were fixed and stained for intracellular p-cofilin. For treatment of resting CD4 T cells with HIV gp120 (IIIB) (Microbix Biosystems Inc., Toronto, Canada), cells were treated with 10 nM gp120 (IIIB) for various times. For treatment with HIV gp120(BAL) (from the NIH AIDS Reagent Program), resting memory CD4 T cells were treated with or without maraviroc (1 μM) (from the NIH AIDS Reagent Program) for 1 hour at 37°C and then treated with HIV gp120(BAL) (100 nM) for various times.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.