Mix fresh EDTA blood with an equal volume of room-temperature PBS in a conical centrifuge tube. #####%%%%% output "Ficoll-Hypaque" are used in line4
{"action": "mix", "output": "Ficoll-Hypaque", "reagent": ["fresh EDTA blood", "room-temperature PBS"], "container": ["conical centrifuge tube"], "temperature": ["room temperature"]}
Slowly layer 3 ml of Ficoll-Hypaque under every 10 ml of the blood/PBS mixture.
{"action": "layer", "output": "", "reagent": ["Ficoll-Hypaque"], "volume": ["3 ml"], "container": ["the blood/PBS mixture"]}
Centrifuge the mixture at 900 x g for 30 minutes at room temperature with no brake.
{"action": "centrifuge", "output": "", "force": ["900 x g"], "time": ["30 minutes"], "temperature": ["room temperature"]}
Carefully remove the upper plasma layer . #####%%%%% output "the upper plasma layer" are used in line12
{"action": "remove", "output": "the upper plasma layer", "device": [""]}
transfer the mononuclear cell layer to a new tube
{"action": "transfer", "output": "", "container": ["the mononuclear cell layer"]}
Wash the cells three times with PBS (3x volume of the cell layer), centrifuging each time for 10 minutes at 400 x g. 6.
{"action": "rinse", "output": "", "reagent": ["PBS"], "times": ["three times"], "volume": ["3x volume of the cell layer"], "force": ["400 x g"], "time": ["10 minutes"]}
Resuspend the cells in complete RPMI-10 for immediate use .
{"action": "resuspend", "output": "", "container": ["the cells"], "reagent": [""], "volume": [""]}