Rat igg2a k isotype control fitc

RPMI 1640 cell culture medium, PBS, and penicillin/streptomycin were obtained from Hyclone (Thermo Scientific, Waltham, Massachusetts, USA). Fetal bovine serum (FBS) was obtained from GIBCO (California, USA). ALA hydrochloride powder was obtained from Shanghai Fudan-Zhangjiang Bio-Pharmaceutical Co, Ltd (Shanghai, China). The following chemicals and commercially available assay kits were used: apoptosis detection kit Annexin-V-FITC/PI (R&D Systems, Minneapolis, MN), rabbit anti-Mouse CD80-FITC, rabbit anti-Mouse CD86-FITC, rabbit anti-Mouse MHC-II-PE, rat IgG2a K Isotype Control FITC, Armenian Hamster IgG Isotype Control PE (eBioscience, USA), Hoechst 33342/PI kit (Beyotime Institute of Biotechnology, China), Mouse IFN-γ, IL-12 and IL-10 ELISA Kit (R&D Systems), and MTT assay Kit (Sigma-Aldrich, St Louis, MO, USA).

Flow cytometry was conducted as previously described (Hou et al., 2016 (link)). The antibodies used in this study were as follows: Anti-Human CD14 PerCP-Cy5.5, mouse IgG1κ isotype control PerCP-Cy5.5, anti-mouse CD11b FITC, rat IgG2a K isotype control FITC, anti-mouse TIM-3 PE, and rat IgG2α K isotype control PE (all obtained from eBioscience, San Diego, CA, USA), anti-human Tim-3-PE, and rat IgG_2A isotope control-PE (both obtained from R&D Systems). The cells were detected and analyzed using a FACS Canto II flow cytometer (BD Biosciences, San Jose, CA, USA), and the gates for positive cells were defined using the isotype controls.

Fluorescence-activated cell sorting (FACS) was used to determine cAMSC immunophenotype. Cells were washed two times with phosphate-buffered saline (PBS; Thermo Fisher Scientific, Waltham, MA, USA) TrypLE™ Express (Gibco, Grand Island, NY, USA) was used to detach the cells. Cells were washed with PBS (Thermo Fisher Scientific) two times, counted, and aliquoted in a 96-well plate (1 × 10⁵ cells/100 µL per well). In each well, 5 µL of fluorochrome-conjugated antibodies or isotype control antibodies with fluorescein isothiocyanate (FITC) or phycoerythrin (PE)—CD29 Monoclonal Antibody-PE (Invitrogen, CA, Carlsbad, USA), CD44 Monoclonal Antibody-FITC, CD90 (Thy-1) Monoclonal Antibody-PE, CD34 Monoclonal Antibody-PE, CD45 Monoclonal Antibody-FITC, Rat IgG2a kappa Isotype Control-FITC, Rat IgG2b kappa Isotype Control-PE, Mouse IgG1 kappa Isotype Control-PE, and Rat IgG2b kappa Isotype Control-FITC (eBioscience, CA, San Diego, USA)—were added to the aliquoted cell suspensions. After 30 min of incubation at 4 °C, the cells were centrifuged (1500 rpm/3 min) and washed with PBS two times. Cells were transferred in round tubes with 5 mL of PBS, analyzed using FACSCalibur™, and Cell Quest software (BD Biosciences, CA, San Jose, USA) was used to calculate CD (Cluster of Differentiation) marker percentages. For each antibody, 10,000 cells were used.