Human cd4 or cd8 t cell isolation kit

Myofibroblasts in the si-TDO2-1, si-TDO2-2, and si-NC groups were then seeded in 96-well plates (10,000 cells per well, 8 wells per group) for 6 hours for total adherence, respectively. For the in vitro TDO2 inhibitor study, the sorted TDO2⁺ and TDO2^– myofibroblasts were added to 96-well plates (10,000 cells per well) for 6 hours for total adherence. The myofibroblasts were divided into 3 groups (6 wells per group). TDO2⁺ myofibroblasts were added to the TDO2⁺ and TDO2⁺ LM10 group, while TDO2^– myofibroblasts were added to the TDO2^– group. The TDO2⁺ LM10 group was treated with the TDO2 inhibitor LM10 (5 μM per plate) (39 (link)), whereas the other 2 groups were treated with an equal volume of solvent. CD3⁺ T cells from PBMCs from 6 healthy donors were isolated by negative selection using magnetic cell separation (MACS) (human CD3⁺ T cell Isolation Kit, Miltenyi Biotec). CD4⁺ and CD8⁺ T cells were isolated from CD3⁺ T cells by positive selection using MACS (human CD4⁺ or CD8⁺ T cell Isolation Kit, Miltenyi Biotec). The isolated CD4⁺ and CD8⁺ T cells were incubated with CFSE for 15 minutes at 37°C before being added to 96-well plates (100,000 T cells per well). The coculture system was then incubated with anti-CD3 and anti-CD28 antibodies (250 ng/mL) and IL-2 (10 ng/mL). Cells were harvested for flow cytometric analyses after 3 days of coculturing.

Peripheral blood mononuclear cells (PBMCs) were prepared from the whole blood of healthy or SLE donors using a density gradient centrifugation method with Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA). Cells were then stained with anti-CD3 (OKT3), anti-CD4 (OKT4), and anti-CD8 (SK1). Some methods required that CD4 or CD8 T cells be isolated from PBMCs using a human CD4 or CD8 T cell isolation kit (Miltenyi Biotec). The cells were then stimulated with pre-coated anti-CD3/28 mAb for 3 days. Intracellular production levels of IFN-γ were measured as described above.

Fresh blood was obtained from healthy volunteers; the written informed consent was obtained. Studies were performed in accordance with the Declaration of Helsinki and were approved by the Research Ethics Board of the Xinhua Hospital, Shanghai Jiao Tong University School of Medicine. Peripheral blood mononuclear cells (PBMCs) were isolated from fresh heparinized blood by standard density gradient centrifugation with Ficoll-Paque Plus (GE Healthcare). CD4⁺ and CD8⁺ T cells were obtained by negative selection using a human CD4⁺ or CD8⁺ T cell isolation kit (Miltenyi) and seeded with 5 × 10⁵ cells per well in 96-well plates. The complete medium (RPMI 1640 with 10% fetal bovine serum) was added to 50 ng/mL recombinant human IL-2 (Peprotech) and cultured for 48 h for the cell activation.
IL-32 shRNA lentivirus (Genechem Company) and activated CD4⁺ and CD8⁺ T cells were mixed according to MOI = 10 plus the infection enhancer B-1 (Genechem Company). The mixture was centrifuged at 1,200 revolutions/min, 30 min at room temperature. After 24 h, the culture medium was half-exchanged. The transfection efficiency was detected by green fluorescent protein fluorescence expression under the microscope and detects mRNA level using real-time polymerase chain reaction (PCR) at 72 h.