Note: All procedures are done in sterile environment in a safety cabinet. #####%%%%% output "mouse" are used in line4
{"action": "note", "output": "mouse", "reagent": [""], "device": ["a safety cabinet"]}
Day 1 Kill mouse by cervical dislocation. #####%%%%% output "alcohol soaked" are used in line6
{"action": "eliminate", "output": "alcohol soaked", "reagent": ["mouse"]}
Spray mouse thoroughly with 70% alcohol () lay down on 70% alcohol soaked paper.
{"action": "spray", "output": "", "reagent": ["70% alcohol"], "device": ["mouse"]}
Make a small incision below the sternum of the mouse .
{"action": "make", "output": "", "length": [""]}
peel the fur back from the chest
{"action": "peel", "output": "", "device": [""]}
Use sterile scissors .
{"action": "use", "output": "", "device": ["sterile scissors"]}
Note: 1:1 mixture of air can also be used to avoid leaking.
{"action": "note", "output": "", "force": [""], "concentration": [""]}
Do not remove the needle.

Give 30 sec massage on two sides of the mouse with fingers.
{"action": "give", "output": "", "device": ["fingers"]}
Collect the PBS with the cells into a Falcon tube with the syringe.
{"action": "collect", "output": "", "container": ["the cells"]}
Note: Needle can be replaced for a bigger one to collect cells from the peritoneal cavity.
{"action": "note", "output": "", "force": ["", ""], "concentration": ["", ""]}
Open the peritoneal cavity with new scissor . #####%%%%% output "the PBS" are used in line26
{"action": "open", "output": "the PBS", "device": ["new scissor"]}
collect the rest of the PBS with a sterile pipette
{"action": "collect", "output": "", "reagent": ["the PBS"]}
Place the Falcon tube onto ice until centrifugation.

Resuspend cells in 5 ml medium [with 10 ng/ml IL-3 per two mice (approximately 1 million cells obtained per mouse)] transfer them into a small culture flask.
{"action": "resuspend", "output": "", "reagent": ["cells"], "volume": ["5 ml medium"]}
Put the flasks into the incubator (37 °C, 5% CO2).
{"action": "put", "output": "", "container": ["the flasks"]}
Day 3 Remove non-adherent cells by discarding the medium .
{"action": "remove", "output": "", "container": ["non-adherent cells"], "volume": [""]}
Put them back into the incubator (37 °C, 5% CO2).
{"action": "put", "output": "", "temperature": ["the incubator (37 \u00b0C, 5% CO2)"]}
Day 6 Add 5 ml fresh medium (with 10 ng/ml IL-3 ) to every flask. #####%%%%% output "Kimura staining" are used in line42
{"action": "add", "output": "Kimura staining", "volume": ["5 ml"], "container": ["every flask"]}
Cell number can be measured with trypan blue staining in haemocytometer.

Purity can be tested with Kimura staining by mixing the cell suspension after 5 min at 37 °C count cells under a light microscope.
{"action": "test", "output": "", "reagent": ["Kimura staining"]}
Note: Expected cell number is 0.5-1.5 x 106 PCMC/mouse after culturing. #####%%%%% output "APC-conjugated" are used in line46
{"action": "note", "output": "APC-conjugated", "time": ["culturing"]}
Purity was assessed on the basis of APC-conjugated c-kit .
{"action": "evaluate", "output": "", "reagent": ["APC-conjugated"]}