Centrifuge the cell suspension at 200 x g at room temperature for 5 min.
{"action": "centrifuge", "output": "", "temperature": ["room temperature"], "time": ["5 min"], "force": ["200 x g"]}
Remove the supernatant . #####%%%%% output "the supernatant" are used in line10
{"action": "remove", "output": "the supernatant", "reagent": [""]}
suspend the cell pellet with 2 ml ACK lysing buffer for 1 min to deplete red blood cells
{"action": "suspend", "output": "", "volume": ["2 ml"], "reagent": ["ACK lysing buffer"], "time": ["1 min"]}
If red blood cells are not completely depleted, repeat the ACK lysing buffer step until they are.
{"action": "repeat", "output": "", "reagent": ["the ACK"]}
Filter the cell suspension through a 40 μm nylon strainer.
{"action": "rinse", "output": "", "container": ["the cell suspension", "a 40 \u03bcm nylon strainer"], "device": [""], "reagent": ["the supernatant"]}
Wash the strainer with 2 ml 1x DPBS for 5 min.
{"action": "wash", "output": "", "container": ["the strainer"], "reagent": ["2 ml 1x DPBS"], "time": ["5 min"]}
Wash the cell pellet with 1x DPBS with 20 ng/ml murine M-CSF in a 100 mm Petri dish.
{"action": "wash", "output": "", "reagent": ["1x DPBS with 20 ng/ml murine M-CSF"], "container": ["a 100 mm Petri dish"]}
suspend in 15 ml complete DMEM medium
{"action": "suspend", "output": "", "volume": ["15 ml"], "reagent": ["complete DMEM medium"]}
Incubate at 37 °C, 5% CO2. #####%%%%% output "fresh complete DMEM medium" are used in line20
{"action": "incubate", "output": "fresh complete DMEM medium", "temperature": ["37 \u00b0C"]}
After 3 days, replace half of the medium with fresh complete DMEM medium.
{"action": "replace", "output": "", "reagent": ["fresh complete DMEM medium"]}
Repeat this step every 2 days.
{"action": "repeat", "output": "", "reagent": ["fresh complete DMEM medium"]}