4.14. IBSP and CRYAB Protein Detection by Flow Cytometry

T-Ti scaffolds were washed three times in PBS and trypsin used to dissociate hMSCs. Cells were fixed and permeabilized and stained at 4 °C for 30 min with rabbit primary unconjugated antibodies against human Bone Sialoprotein and human Alpha B Crystallin. After two washes in ice-cold FACS buffer (PBS, 2% FBS, 0.1% NaN₃), cells were stained with Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (all antibodies from abcam) at 4 °C for 30 min in the dark. After a final wash, data were acquired with a CytoFLEX flow cytometer (Beckman Coulter), collecting a minimum of 30,000 events and using Anti-Rabbit IgG H&L stained samples as negative controls.