Anti mouse ly 6g and ly 6c particles dm

MDSCs were isolated from the spleen and tumor tissue from L-4F/Sc-4F treated mouse using anti-mouse Ly-6G and Ly-6C particles-DM (BD Biosciences, USA). The purity of each cell population was > 99%. The immunocytes from spleen (splenocytes) from C57BL/6 mice were isolated. A total of 2×10⁵/well splenocytes were stimulated with coated 6 µg/ml anti-CD3 (BD Biosciences, USA) and 6 µg/ml soluble anti-CD28 mAbs (BD Biosciences, USA) for 3 d and co-cultured at a 4:1 ratio with sorted MDSCs in 96-well flat-bottom plates. After 3 d, the cells were stained with anti-CD3-PerCP, anti-CD4-APC-H7, and anti-CD8-APC. Then the CD3⁺CD4⁺ and CD3⁺CD8⁺ lymphocytes was analyzed.
The culture supernatants were collected to detect the concentration of IFN-γ (interferon-γ) using an mouse IFN-γ ELISA assay kit (BD Biosciences, USA) and TNF-β using an mouse TNF-β ELISA assay kit (BioLegend, USA) according to the manufacturer’s protocol. Each experiment was performed in triplicate.
The culture medium consisted of RPMI 1640 medium supplemented with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (0.1 mg/ml), 2-ME (50 μ M), and 10% heat-inactivated FBS.

As described above, mouse MDSCs were induced in vitro. Then induced MDSC cells were sorted using anti-mouse Ly-6G and Ly-6C particles-DM (BD Biosciences, USA). Ly6G⁺Ly6C⁺ cells (MDSC) proliferation and cell division was assayed by using carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen, USA) labeling. Cell apoptosis was assessed using a MitoScreen (JC-1) assay kit (BD Biosciences, USA) according to the manufacturer’s protocol. The labeled cells were treated with vehicle or the different concentration of L-4F (5, 10, 20 μg/ml) for 48 h. Then, the cells were assessed using BD FACSVerse flow cytometer and quantified with FlowJo 7.6 software.

MDSCs were isolated from the spleen and tumor tissue from L-4F/Sc-4F treated mouse using anti-mouse Ly-6G and Ly-6C particles-DM (BD Biosciences, USA). The purity of each cell population was > 99%. ROS activity was detected by using a fluorometric intracellular ROS kit (Sigma-Aldrich, USA) and the concentration of H₂O₂ was detected by using an Intracellular hydrogen peroxide assay kit (Sigma-Aldrich, USA) according to the manufacturer’s protocol. Mean fluorescence intensity (MFI) was analyzed using FlowJo 7.6 software (BD Biosciences, USA).