Remove medium to one 15ml tube 3ml/plate 1xPBS wash once 0.5ml/plate 1X trypsin addition room temperature, 10 min adding original FBS/DMEM medium to stop the protease reaction combine the cell suspension in one 15ml tube centrifuge 1000 rpm, 3 min remove supernatant resuspend in 1ml 1xPBS (wash again) transfer to one 1.5ml tube centrifuge 2500 rpm, 3 min remove supernatant