Rat igg2b k isotype control fitc

Nicotine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Monoclonal antibodies (anti-mouse CD3-FITC, anti-mouse CD4-APC, anti-mouse CD8-PE-cy7, Rat IgG2b K Isotype Control FITC, Rat IgG2b K Isotype Control APC and Rat IgG2a K Isotype Control PE-cy7) and Annexin V PE Apoptosis Detection kit were purchased from eBioscience (San Diego, USA). Anti-mouse CD95-PE-cy7 was obtained from BD Biosciences (New Jersey, USA). Mouse IgG1 and IgG2a ELISA kits were obtained from MultiSciences (Hangzhou, Zhejiang, China). Mouse IL-4 ELISA kits were obtained from Dakewe Biotech (Shenzhen, Guangdong, China). Trizol was purchased from Life Technologies (Gaithersburg, MD, USA). Reverse transcription and RT-qPCR kits were purchased from TaKaRa Biotechnology (Dalian, Liaoning, China). All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). All chemicals and reagents were analytical grade.

Nicotine and α-bungarotoxin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Monoclonal antibodies (anti-mouse CD3-FITC, anti-mouse CD4-APC, anti-mouse CD8-PE-cy7, Rat IgG2b K Isotype Control FITC, Rat IgG2b K Isotype Control APC and Rat IgG2a K Isotype Control PE-cy7) and Annexin V/PE Apoptosis Detection kit were purchased from eBioscience (San Diego, USA). Anti-mouse CD95-PE-cy7 was obtained from BD Biosciences (New Jersey, USA). Mouse IgG1 and IgG2a ELISA kits were obtained from MultiSciences (Hangzhou, Zhejiang, China). Mouse IL-4 ELISA kit was obtained from Dakewe Biotech (Shenzhen, Guangdong, China). Anti-a7 nAChR and anti-TET2 were obtained from Abcam (San Diego, USA). Trizol was purchased from Life Technologies (Gaithersburg, MD, USA). Reverse transcription and RT-qPCR kits were purchased from TaKaRa Biotechnology (Dalian, Liaoning, China). RPMI-1640 medium and fetal bovine serum (FBS) were purchased from HyClone (Logan, UT, USA). Caspase-3 Action Detection kit and Caspase-8 Action Detection kit were from Beyotime Biotechnology (Shanghai, China). The TET2 siRNA and all primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The TIANamp Genomic DNA Kit was from TIANGEN Biotech Co., Ltd. (Beijing, China). All chemicals and reagents were analytical grade.

SCs were isolated from 8-week-old C57BL/6 femur and tibia bone marrows, as previously described [10 ]. Cells were used in the study between the third and fifth passages. For flow cytometry characterization, MSCs were harvested with Accutase (Stemcell Technology, Vancouver, BC, Canada), stained, and immediately acquired on a FACSCalibur system (BD Biosciences Pharmingen, San Diego, CA). The following monoclonal antibodies were used: anti-mouse CD29-PE (eBioscience, San Diego, CA), CD45 PE (Biolegend, San Diego, CA, USA), CD11b FITC (Biolegend, San Diego, CA), CD14 PE (BD Biosciences Pharmingen, San Diego, CA), rat anti-mouse CD90 purified (eBioscience, San Diego, CA) and anti-rat Alexa Fluor 488 (Invitrogen, Waltham, MA), rat IgG2b k Isotype Control FITC (eBioscience, San Diego, CA), rat IgG1 k Isotype Control PE (eBioscience San Diego, CA), and rat IgG2b k Isotype Control PE (eBioscience, San Diego, CA). Data were analyzed using the FlowJo software, version 10.5.3 (Becton Dickinson and Company, Franklin Lakes, NJ).