Pe anti human cd4 antibodies

In order to study the effect of S3-15 on the immune system, after 4 weeks of S3-15 treatment, orbital venous blood with EDTA-anticoagulated was collected for T lymphocyte subsets analysis in osteoporosis mice. We used flow cytometric to determinate T lymphocyte subsets according to the guidelines for Flow Cytometric of Beckman Coulter, FC500. Cells were stained according to manufacturer’s instruction of PE-Cyanine7 anti-Human CD8a and PE anti-Human CD4 antibodies (Tonbo Biosciences). Data analyzing was performed by using CXP software (Beckman Coulter, Supplementary Fig. 9).

T lymphocyte cell proliferation assay was performed according to a reported method^{58 (link)} with some modification. Briefly, 5 × 10⁶ cells /mL cells were stained 2.5 μM CFSE (Tonbo Biosciences) and incubated at 37 °C for 12 min. Then washed and discarded the supernatant and adjusted the CFSE stained cell to 1.75 × 10⁶ cells /mL. Cells were diluted in 1640 completed medium containing 5 μg/mL anti-CD3 antibody (Tonbo Biosciences) to 3 × 10⁵ cells/mL and seed in a 96-well plate or 24-well plate that pre-packaged 0.4 μg/well anti-CD28 antibody and 1.6 μg anti-CD28 antibody (24-well) (Tonbo Biosciences). Cells were then cultured for 3 days, 9 days or 12 days. Subsequently, the proportion of CD4⁺ and CD8⁺ cells was determined by flow cytometry (Beckman Coulter, FC500) according to manufacturer’s instruction of PE-Cyanine7 anti-Human CD8a and PE anti-Human CD4 antibodies (Tonbo Biosciences). Data analyzing was performed by using CXP software (Beckman Coulter, Supplementary Fig. 8).