Human cd4 t cell isolation kit

Peripheral blood mononuclear cells were isolated from HCs and patients with SLE using human peripheral blood lymphocyte separation medium (Tianjin Hao Yang Biological Manufacture, Tianjin, China) within 6 h of sample collection. Peripheral blood CD4⁺ T cells were then purified following immunomagnetic separation using a human CD4⁺ T cell isolation kit (BD Biosciences, San Jose, CA, United States) according to the manufacturer’s instructions. The isolated human primary CD4⁺ T cells were lysed in TRIzol reagent (Invitrogen Life Technologies, Grand Island, NY, United States) for RNA purification. The isolated RNAs were digested by DNase I (Invitrogen, Waltham, MA, United States) to remove residual DNA, then collected in 25 μL of DNase/RNase-free water. Isolated RNA was stored at −80°C for use.

Peripheral blood mononuclear cell (PBMCs) were isolated from HCs and SLE patients using human peripheral blood lymphocyte separation medium (Tianjin HaoYang Biological Manufacture, Tianjin, China) within 6 h of sample collection. CD4⁺ T cells were then purified following immunomagnetic separation using a human CD4⁺ T cell isolation kit (BD Biosciences, San Jose, CA, United States) according to the manufacturer’s instructions. For RNA purification, the isolated human CD4⁺ T cells were lysed in TRIzol reagent (Invitrogen Life Technologies, Grand Island, NY, United States). The extracted RNA was further digested by DNase I (Invitrogen, Waltham, MA, United States) to remove residual DNA, and subsequently separated from each sample using TRIzol reagent/RNeasy Mini kit (Qiagen, Hilden, Germany). Total extracted RNA was stored for use at −80°C.

The PBMCs were isolated from healthy human donors through Ficoll gradient centrifugation, followed by culturing in the conditioned RPMI 1640 medium. Human primary CD4⁺ T lymphocytes were then purified with a human CD4⁺ T-cell isolation kit according to the manufacturer's instructions (BD Biosciences). The isolated human primary CD4⁺ T lymphocytes were then maintained in the conditioned RPMI 1640 medium (Hyclone) and stimulated with phytohaemagglutinin (5 ng ml⁻¹, Roche Applied Science) and interleukin-2 (IL-2, 10 ng ml⁻¹, R&D Systems), or anti-CD3 antibody (1 μg ml⁻¹, BD Biosciences), anti-CD28 antibody (1 μg ml⁻¹, BD Biosciences) and IL-2 (10 ng ml⁻¹, R&D Systems) for 48 h. Then, the cells were washed three times with PBS buffer, and were cultured in the presence of IL-2 (10 ng ml⁻¹).