CCR5 tropic HIV-1 inhibition ex vivo. (A) Splenocytes were isolated from a transplanted mouse at 20 weeks after CD34⁺ cell injection. Cells were activated with PHA for 2 days and interleukin-2 for 5 days. CD8⁺ cells were depleted and sorted for EGFP⁺ and mCherry⁺ cells at 99.6% purities. Sorted cells (4 × 10⁴) were infected with CCR5 tropic HIV-1_{NFNSX SL9} or CXCR4 tropic HIV-1_NL4-3 for 2 hours at MOI of 2.5 in parallel and in triplicate. Cells were washed 3 times after the infection. The amount of remaining input HIV-1 particles in culture supernatant was monitored 1 hour after infection by HIV p24 enzyme-linked immunosorbent assay. The amount of HIV production in culture supernatant was monitored by HIV p24 enzyme-linked immunosorbent assay at days 4, 7, and 12 after infection during the culture. The average p24 production in culture supernatant. Error bar represents SD. shRNA significantly affected HIV growth curve of HIV-1_{NFNSX SL9} but not HIV-1_NL4-3 (P < .001 and P = .38, respectively, 2-way analysis of variance). (B) CCR5 expression in EGFP⁺ and mCherry⁺ cells at 12 days after HIV-1 infection.