Pe conjugated anti human cd4 antibody

To detect the expression of CCR5 in cells edited by CRISPR/SaCas9, we collected the control-modified and CCR5-sgRNA/SaCas9 modified TZM-bl cells 3 days post transfection and washed with cold 1 × PBS for three times. Then the cells were strained with APC conjugated anti-human CCR5 antibody (Biolegend) for 30 min on ice and analyzed by flow cytometry (FACS AriaIII, BD). To count the number of CD4⁺ T cells in blood obtained from the mice in different time points, we firstly treated the blood samples by red blood lysis buffer (BD Biosciences) for 15 min, then stained the samples with PE conjugated anti-human CD4 antibody (BD Biosciences), APC conjugated anti-human CD3 antibody (BD Biosciences) and FITC conjugated anti-human CD8 antibody (BD Biosciences) for 30 min on ice. At last, all samples were analyzed by flow cytometry (FACS AriaIII, BD) and Flow Jo software (Treestar). For western blotting assay, we lysed the cells with lysis buffer containing 50 mM Tris-HCl pH = 7.4, 1% Triton X-100, 150 mM NaCl, 0.1% SDS, 1.5 Mm EDTA, 0.25% deoxycholate and protease inhibitor PMSF and cocktail (Roche Applied Science) on ice for 30 min. The lysates were centrifuged by 12,000 rpm for 10 min and the supernatants were mixed with 2 × SDS loading buffer incubation at 100 °C for 10 min. The proteins were detected by SDS-PAGE with anti-GAPDH (Proteintech) and anti-CCR5 antibodies (Proteintech).

We isolated CD4⁺ T helper cells from PBMCs according to the manufacturer’s instructions (https://www.miltenyibiotec.com/_Resources/Persistent/3c804fa07b66b63215bbacbf43387804b151d77f/SP_CD4.pdf). Venous blood samples were collected from the patients with ASO or the healthy donors at the First Affiliated Hospital of Sun Yat-sen University, and PBMCs were isolated through Ficoll centrifugation (GE Healthcare, Catalog #17144002). CD14⁺ cells were isolated from PBMCs with CD14 magnetic bead kits (Miltenyi, Catalog# 130-050-201) and FITC-conjugated anti-human CD14 antibody (BD, Catalog# 555397) according to the manufacturer’s instructions. CD4⁺ T cells were isolated from the rest of the CD14⁻ cell suspensions by positive selection with a CD4 magnetic bead kit (Miltenyi, Catalog# 130-045-101) and PE-conjugated anti-human CD4 antibody (BD, Catalog# 555347) according to the manufacturer’s instructions. Isolated CD4⁺ T cells, CD14⁺ cells and CD4⁻CD14⁻ cells were validated by FACS and frozen in liquid nitrogen for future experiments.