Dynabeads flowcomp human cd4 kit

Cell-associated HIV-1 proviral DNA was measured at baseline and week 4, and cell-associated HIV-1 RNA was measured at each visit through week 16 (baseline, days 2, 4, 8, week 4 and 16). CD4⁺T cells were isolated from PBMCs using Dynabeads FlowComp Human CD4 Kit (Invitrogen). DNA and RNA isolation was performed with AllPrep DNA/RNA Mini Kit (Qiagen). Isolated RNA was treated with DNase I, Amplification Grade (Invitrogen) and purified with RNeasy Mini Kit (Qiagen). Real time PCR was performed with AmpliTaq Gold with Buffer A or AmpliTaq Gold DNA Polymerase with Buffer II and MgCl2 with ROX Reference Dye (Applied Biosystems) using the Stratagene Mx3000P QPCR System (Agilent Technologies). PCR reactions were performed in quadruplicate. Note that for each case an optimal primers/probe set was determined, which gave the highest amplification efficiency among three primer/probe sets tested. Accordingly two primer/probe sets were used, RF/RR/PB (41 (link)) for 9 participants and 6F/84R/HIV gag probe (42 (link)) for 6 participants.
Cell-associated RNA was primed with random hexamers and reverse transcribed with Superscript II (Invitrogen). PCR reagent mix containing primers, probe, and AmpliTaq Gold was added to each of RT product, and quantitative rtPCR was performed as described above in quadruplicate. No reverse transcriptase control was also included.

CD4+ T cells were isolated from PBMCs by the Dynabeads FlowComp Human CD4 kit (Invitrogen). Purity was consistently >95% of live cells as determined by CD3 and CD4 staining by flow cytometry. CD4+ T cells were grown in 96-well plates or anti-CD3 coated 96-well plates (BD) with or without 20 μM minocycline hydrochloride (Sigma) in 100 μl RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 1 mM HEPES buffer, and 2 mg/mL gentamicin. After 24 hours, a 50 μL aliquot containing 60 μM minocycline was added to replenish minocycline at a final concentration of 20 μM in the 150 μL well volume. Some wells were also stimulated with 1,000 U/mL each of IFNα-2a and IFNβ-1a (PBL). All wells had a final volume of 150 μL and were cultured for an additional 24 hours (total 48 hours) before flow cytometry analysis.