Annexin 5 detection kit

Three experimental groups as well as the control were centrifuged. After centrifuging the adipose tissues for 3 min at 1610 x g, 10 mL of adipose tissue from the middle layer of each sample group was collected, and Type II collagenase solution was added and mixed with each sample group for the enzymatic digestion. After histolysis for 1 h in an incubator at 37°C, the treated tissues were centrifuged for 10 min at 1610 x g and the supernatant was removed. All sample groups were mixed with 2 mL of Dulbecco's modified Eagle's medium (DMEM; Gibco, Thermo Fisher Scientific). Apoptotic effects of freezing methods were examined by the Annexin V Detection Kit (Abcam, Cambridge, UK) in accordance with the protocol of manufacture. Samples were incubated with Annexin V fluorescein isothiocyanate and propidium iodide (PI) phycoerythrin (PE) conjugated antibodies. Detections were performed using FACS Calibur flow cytometry (BD Biosciences, San Jose, CA). Analysis of the data was executed via the CellQuest Pro program (BD Biosciences).^{11 (link)}

FLS grown on soft dishes at 2 kPa (ExCellness) for 72 hours coated with fibronectin with various stimuli for 48 hours were collected by trypsination. RA FLS were incubated in 1X annexin V binding buffer with Annexin V detection kit (ab14155, Abcam) or permeabilized and fixed before 1h labeling with ki67 antibody. Data were acquired using a FACSCanto II cytometer and analyzed with the BD FACS Diva software 6.1.3 (BD Nova Biosciences, UK).