Collect ~200 ml culture medium.
{"action": "collect", "output": "", "volume": ["200 ml"], "container": ["culture medium"]}
, centrifuge at 1,000 g for 10 min at 4°C to remove intact cells #####%%%%% output "crude exosome" are used in line6
{"action": "centrifuge", "output": "crude exosome", "speed": ["1,000 g"], "temperature": ["4\u00b0C"], "reagent": ["intact cells"], "time": ["10 min"]}
Prepare 10-90% sucrose solutions, mix crude exosome preparation with 90% sucrose solution to 82% final concentration, layer remaining sucrose solutions from 70% to 10% on top, for 16 hr.
{"action": "prepare", "output": "", "concentration": ["10-90% sucrose solutions"], "reagent": ["crude exosome"]}
Collect 2 ml fractions, .
{"action": "collect", "output": "", "volume": ["2 ml fractions"]}
add 9 ml PBS to each,
{"action": "add", "output": "", "volume": ["9 ml PBS"], "mass": [""]}
centrifuge at 100,000 g for 1 hr, resuspend pellets in 50 μl PBS
{"action": "centrifuge", "output": "", "speed": ["100,000 g"], "time": ["1 hr,"], "volume": ["50 \u03bcl"]}
The third fraction (34%-40% interface) contains exosomes, the sixth fraction (70%-82% interface) contains aggregates. #####%%%%% output "exosomes" are not used
{"action": "include", "output": "exosomes", "reagent": [""]}