Ficoll paque premium

HEK293T cells, Ghost-CXCR4 (X4) cells, Jurkat T cells and HIV-1 virus were used as described previously47 (link). Whole blood samples from healthy donors were purchased from the Wuhan Blood Center (Wuhan, China). The human peripheral blood mononuclear cells (PBMCs) were separated from the whole blood by centrifugation with Ficoll-Paque Premium (BD). The primary human CD4⁺ T cells were further purified and enriched by the CD4⁺ T cell isolation Kit (Miltenyi Biotech) according to the manufacturer’s instructions and then maintained in complete RPMI medium supplemented with 10% FBS. Before transfection, the primary human CD4⁺ T cells were stimulated 2 to 3 days with anti-CD3/anti-CD28-coated plate in the presence of recombinant human interleukin-2 (20 IU/ml, Roche Applied Science). The PBMCs of whole blood from healthy Rhesus macaque (grown in the Center for Animal Experiment and ABSL-3 Laboratory, Wuhan University, China) were isolated by centrifugation with Ficoll-Paque Premium (BD). The CD4⁺ T cells were further purified with a non-human primate CD4⁺ T cell selection kit (Miltenyi Biotech). The cells were then activated with anti-CD3 (clone FN-18)/anti-CD28 (clone L293) (Invitrogen) coated on culture plate for 3 days.

TZM-bl cells, Jurkat T cells and human CD4⁺ T cells were cultured and prepared as previously described [37 (link)]. The human blood samples for primary CD4⁺ T isolation were taken from healthy donors in Wuhan Blood Center (Wuhan, China), and the peripheral blood mononuclear cells (PBMC) were isolated with lymphocyte separation medium Ficoll-paque Premium (BD). The primary CD4⁺ T cells in PBMC were separated and enriched using a human CD4⁺ T cell isolation kit (Miltenyi Biotech), according to the manufacturer’s instructions. Primary CD4⁺ T cells were cultured in RPMI 1640 medium and stimulated by CD3/CD28 in the presence of recombinant human interleukin-2 (IL-2, 10 IU/ml) for 3 days.