Mouse na ve cd4 t cells isolation kit

Mice were subjected to cervical dislocation following anesthesia with isoflurane. The mice were then sterilized in 70% ethanol for 5 min. The spleen was dissected by aseptic operation and homogenized. The crushed spleen tissue was resuspended in PBS and filtered through a 70‐µm cell strainer. Red blood cells (RBCs) were lysed by incubating the cells in RBC lysis buffer (Solarbio, #R1010) for 15 min at 4 °C. Splenocytes were then collected by centrifugation at 1500 rpm for 5 min. For the isolation of naïve CD4+ T cells in the spleen, a Mouse Naïve CD4+ T Cells Isolation Kit (Miltenyi Biotec, #130‐104‐453) was used according to the manufacturer's protocol. The obtained splenocytes were incubated with a cocktail of biotinylated negative selection antibodies and then subjected to magnetic labeling with anti‐biotin microbeads. The magnetically labeled cells were absorbed using a Separation Column in a magnetic field, and naïve CD4+ T cells were collected for subsequent experiments.

Naïve CD4⁺ T cells were purified from the splenocytes of 6–8-week-old MRL and MRL-lpr mice by negative selection using a mouse naïve CD4⁺T cells isolation kit from Miltenyi Biotec. The purity of naïve CD4⁺ T cells was determined by staining the cells with surface maker CD4 and CD44 and characterized as CD4⁺CD44⁻. The naïve CD4 T cells were transfected with EGR2 and NC DsiRNA. Twenty-four hours after DsiRNA transfection, the transfected naïve CD4⁺ T cells were cultured at Th1 polarization condition (plate-bound anti-CD3 (2 μg/ml), anti-CD28(1 μg/ml, IL-2 (5 ng/ml), IL-12 (10 ng/ml) and anti-IL4 (10 μg/ml)) for 3 days. The culture supernatant was collected for IFNγ enzyme-linked immunosorbent assay (ELISA). The cells were stimulated with PMA (50 ng/ml), ionomycin (1 μg/ml) and BD GolgiPlug protein transporter inhibitor for 5 h for intracellular flow cytometry analysis.

Naïve CD4⁺ T cells were purified from the splenocytes of 6-7-week-old EGR2^-/-B6/lpr and control mice by using a mouse naïve CD4⁺ T cells isolation kit from Miltenyi Biotec as we previously reported (16 (link)). For Th1 differentiation, naïve CD4⁺ T cells (1.5x10⁶) were plated in 48-well plate and cultured with 2 μg/ml plate-bound anti-CD3 (clone 145-2C11, Bio X cell, Lebanon, NH, USA), 1 μg/ml soluble anti-CD28 (clone 37.51, Bio X cell), 5ng/ml IL-2 (eBioscience), 10ng/ml IL-12 (eBioscience) and 10 μg/ml anti-IL4 (Bio X cell) for 3 days. For Th17 differentiation, naïve CD4⁺ T cells (1.5x10⁶) were plated in 48-well plate and cultured with 2 μg/ml plate-bound anti-CD3, 1 μg/ml soluble anti-CD28, 20ng/ml IL-6, 3ng/ml hTGFβ1, 10 μg/ml anti-IFNγ (Bio X cell) and 10 μg/ml anti-IL4 for 5 days. The differentiated cells were stimulated with PMA (50 ng/ml), ionomycin (1 μg/ml) plus protein transporter inhibitor (BD GolgiPlug) for 5 hours, and then collected for intracellular flow cytometry analysis of IFNγ- and IL-17- expressing cells.