Magi-CCRS cells grown in DMEM, 10% FCS, neomycin and hygromycin. HeLa-CD4 cells grown in same media but without hygromycin. H9 and ACH-2 lines grown in RPMI 1640,and 10% FCS.
Blasts of CD4+T cells were made from blood lymphocytes harvested by immunomagnetic selection using Miltenyi beads (Miltenyi Biotech).
siRNAs were synthesized by Dharmacon Research (Colorado) using 2'ACE protection chemistry. They were CD4, p24, GFP, HPRT, and CD19.
Transfection of siRNA into either attached cells or suspension cells was carried out using oligofectamine (Invitrogen)in DMEM media.
Monoclonal antibodies phycoerythrin-conjugated alpha CD4 and RD-1-conjugated alphaHIV-1 p24 were used for staining.
Total RNA obtained (RNAeasy, Qiagen) for Northern blot analysis. Northern Max protocol (Ambion) was followed.