Anti cd8 pe antibodies

For lymphocytes differentiation, blood samples were harvested at indicated time points and monocytes were separated though isodensity centrifugation. Thereafter, approximately 10⁶ cells were re suspended in 100 μL of PBS and incubated with 2 μg anti-CD3c FITC together with anti-CD4 PE or anti-CD8 PE antibodies (all from BioLegend, San Diego, CA, USA) for 20 min on ice. Thereafter, the cells were washed twice with PBS and flow cytometry was performed using a FACSort instrument (BD Biosciences, San Jose, CA, USA). Data was analyzed using FlowJo software (Tree Star, San Caros, CA, USA).

Bergenin (purity of 95%) was obtained from Meilun Biological Products Co. Ltd. (Dalian, China). The chemical structure of bergenin is shown in Figure 6. The cell culture products were from Gibco BRL (Grand Island, NY, USA). Injectable cyclophosphamide was obtained from Jiangsu Hengrui Medicine Co. (Lianyungang, Jiangsu, China). A HE staining kit was purchased from Beyotime (Jiangsu, China). Concanavalin A (Con A), lipopolysaccharide (LPS) and neutral red (N7005) were provided by Sigma-Aldrich (St. Louis, MO, USA). The Ig kit and cytokines ELISA kits were purchased from Lianke Biotechnology Co. Ltd. (Hangzhou, China). SOD, CAT, GSH-Px and MDA detection kits were provided by Nanjing Jiancheng Bio-engineering Institute (Nanjing, China). 2-(2-Methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, mono- sodium salt] (WST-8) was purchased from Amresco Co. (Solon, OH, USA) Anti-CD4⁺ (APC) and anti-CD8⁺ (PE) antibodies were provided by Bio Legend, Inc. (San Diego, CA, USA).

Splenocytes were resuspended at 10⁶ cells/ml in 10% FBS in PBS and stained with carboxyfluorescein succinimidyl ester (CFSE) (Thermo Fisher) by mixing equal volumes of cell suspension with 0.010 mM CFSE in 10% FBS in 1× PBS for 5 min at 37 °C. Cells were then washed three times with R10 complete medium and incubated for 96 h in the presence of medium alone or 0.010 mg/ml SARS-CoV-2 antigens N or S1 (NUN-C5227 and SIN-C52H4, Acro Biosystems). After 96 h, cells were washed with 0.2 ml 10% FBS in PBS and centrifuged at 300g for 5 min 2at RT. Cells were then stained with anti-CD3-APC (Thermo Fisher, #17-0032-82), anti-CD4-PerCP-Cy5.5 (Biolegend, #100433), or anti-CD8-PE antibodies (Biolegend, #MCD0801) for 30 min at 4 °C. The stained cells were washed twice with 0.2 ml PBS and analyzed by flow cytometry using a FACS Canto II (BD Biosciences) gated first for CD3+ T-cells and then for CD4+/CD8− or CD8+/CD4− populations. Data was analyzed using FlowJo 10 software (FlowJo LLC).