Anti cd29 pe

Tween-80 was purchased from Nanjing Well Pharmaceutical co., LTD. (Nanjing, China). Ethanol (AR grade), phosphate acid (AR grade), ethyl acetate (AR grade), hydrochloric acid (AR grade), n-hexane (AR grade) were all purchased from Sinopharm Chemical Reagent Co. LTD. (Shanghai, China). Standards of ginkgolide A ≥ 95%, ginkgolide B ≥ 95%, ginkgolide C ≥ 95%, ginkgolide J ≥ 95%, bilobalide ≥ 98%, quercetin ≥ 98%, isorhamnetin ≥ 98%, kaempferol ≥ 98% were purchased from National Institutes for Food and Drug Control (Beijing, China). Formic acid (MS grade), acetonitrile (HPLC grade), triffuoroacetic acid (HPLC grade), tetrahydrofuran (HPLC grade) and methanol (HPLC grade) were acquired from Merck (Darmstadt, Germany). Fetal Bovine Serum (FBS) was obtained from GIBCO (Australia Origin). DME/F12, PBS buffer, Trypsin, and Penicillin-streptomycin were purchased from Hyclone (Logan, Utah, United States). Anti-CD90-PerCP, anti-CD45-FITC, anti-CD29-PE and anti-CD34-Alexa Fluor 647 were acquired from BD Biosciences (San Diego, United States).

For identification of immunophenotypes, aging ASCs and DFAT cells were harvested at P4. Cells were trypsinized, and viability was assessed using 0.4% Trypan Blue (Gibco) and found to be greater than 95%. Then, cells were separated into tubes containing 5 × 10⁵ cells each. The following mouse monoclonal anti-human antibodies conjugated with fluorescein isothiocyanate-conjugated (FITC) and phycoerythrin (PE) were used: anti-CD29-PE, anti-CD31-PE, anti-CD73-PE, anti-CD90-PE, anti-CD105-PE, anti-CD106-PE, anti-CD146-PE (all from BD Biosciences, CA, USA): anti-CD34-FITC, and anti-CD44-FITC (Beckman coulter Inc.); and immunoglobulin G1 isotype control (BD Biosciences). Each aliquot was incubated in the dark at 4 °C for 20 min. Then, cell pellets were washed with PBS and resuspended in 0.1% bovine serum albumin (BSA)/PBS. Flow cytometric data were analyzed with Guava Express Plus version 5.3 software (Guava Technology).