Cd4 t cell negative selection kit

Epidermal mononuclear phagocytes were isolated from abdominal skin using collagenase digestion and LCs, CD11c^hiDCs and CD33^low cells sorted by FACS. A minimum of 3 × 10⁴ of each cell population was then infected with HIV_BaL at MOI = 1 for 2 h and the virus washed off using 3x PBS washes. Infected cells were then cultured human skin fibroblast conditioned media to enhance cell survival^{15 (link)} and JLTR CD4 T cells (which express GFP under control of the HIV-1 promotor^{10 (link)} were added at ratio of 4:1 after 96 h and co-cultured for a further 96 h. The percentage of GFP+ JLTR cells was then determined by flow cytometry. Transfer assays with HIV_Z3678M were performed as above, except CD4 T cells isolated (CD4 T cell negative selection kit, Stemcell) from PBMCs activated for 3 days with PHA (5 µg/mL) and IL-2 (150 IU/mL, Peprotech) were added at a ratio of 2:1 and co-cultured for a further 72 h prior to staining with Live/Dead Near-IR and intracellularly for P24 (KC57)-PE. For Inhibition experiments, sorted MNPS were pre-treated for 1 h with 10 µM Maraviroc (kindly gifted by Paul Gorry, Melbourne), HIV was added for 2 h, cells were washed and cultured for a further 96 h in the presence of maraviroc. At 96 h, maraviroc was washed off prior to the addition of JLTR cells for a further 96 h.

6-week-old female C57BL/6 mice were euthanized, and CD4⁺ T cells were isolated from spleens and lymph nodes using a CD4⁺ T cell negative selection kit (StemCell Technologies, INC). Isolated CD4⁺ T cells in RPMI media were seeded on anti-CD44 antibody-coated glass coverslips mounted in Chamlide chambers. The protein solution was then added to the chamber, and time-lapse imaging was initiated. Differential interference contrast (DIC) and GFP images were recorded at 5-min intervals for 2 h. The acquired time-lapse images were analysed using MetaMorph or ImageJ software 1.48v.