Grow Burkitt’s Lymphoma cell lines from 1 x 10^7 (in one 10 cm dish) to 2 x 10^8 cells (in twenty 10 cm dishes) in 200 mL RPMI 1640 medium containing 10% exosome-depleted FBS in the 5% CO2 incubator at 37 °C.
{"action": "grow", "output": "", "container": ["one 10 cm dish", "twenty 10 cm dishes"]}
The exosome fraction usually locates around the 6th fraction from the top.
{"action": "locate", "output": "", "length": [""]}
Collect the band (about 1 mL) carefully with an autopipette. #####%%%%% output "the exosome fraction (4 µg each)" are used in line10
{"action": "collect", "output": "the exosome fraction (4 \u00b5g each)", "volume": ["about 1 mL"]}
Determine the total protein concentration in the fractions by the Bradford protein assay.
{"action": "determine", "output": "", "reagent": ["fractions"]}
Confirm the exosome fraction (4 µg each) by western blot analysis with anti-CD63 monoclonal antibody (1:1,000 dilution). #####%%%%% output "exosomes" are used in line12
{"action": "confirm", "output": "exosomes", "reagent": ["the exosome fraction (4 \u00b5g each)", "anti-CD63 monoclonal antibody (1:1,000 dilution)"]}
Confirm the efficiency of labeling with a small amount of exosomes under a fluorescent laser scanning microscope.
{"action": "confirm", "output": "", "reagent": ["exosomes"], "device": ["a fluorescent laser scanning microscope"]}