Rat igg2b k isotype control apc

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.

After cell cultivation, cells were dissociated with Accumax (Innovative Cell Technologies, San Diego, CA). Dissociated cells were then stained with Fixable Viability Dye eFluor 450 (eBioscience, San Diego, CA) to distinguish between living and dead cells. For flow cytometric analysis, the cells were incubated with the following fluorescence-conjugated antibodies: anti-CD44 APC (eBioscience), anti-CD24 APC (eBioscience), or anti-CD90 FITC (Miltenyi Biotec, Bergisch Gladbach, Germany). Rat IgG2b, k isotype control APC (eBioscience), mouse IgG1 APC isotype control (R&D Systems), and mouse IgG1 FITC isotype control (R&D Systems) were used as negative controls, respectively. For CD44 variant staining, anti-CD44v9 (Cosmo Bio, Tokyo, Japan) and mouse anti-rat IgG FITC (eBioscience) were used as primary and secondary antibodies, respectively. Rat IgG2a, k Isotype Control (eBioscience) was used as a negative control for the anti-CD44v9 antibody. Flow cytometric analysis was performed using a MACSQuant analyzer (Miltenyi Biotec).
Cell cycle distribution was analyzed with propidium iodide staining followed by flow cytometry. Cells were fixed with 70% ethanol and then resuspended in PI/RNase Staining Buffer (BD Biosciences, Franklin Lakes, NJ). The DNA content of cells was analyzed using a MACSQuant analyzer.

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.