Dissect Lewis Lung Cancer (LLC) tumors (tumor volume ~1,000 : 0.5~0.8 g/tumor) grown on mice. #####%%%%% output "one tumor" are used in line4
{"action": "grow", "output": "one tumor", "reagent": ["Lewis Lung Cancer (LLC)"]}
Place one tumor into a 100 mm Petri dish .
{"action": "place", "output": "", "reagent": ["one tumor"], "container": ["a 100 mm Petri dish"]}
add 5-10 ml HBSS at room temperature (RT) #####%%%%% output "tumors" are used in line8
{"action": "add", "output": "tumors", "volume": ["5-10 ml HBSS"], "temperature": ["RT"]}
Quickly mince tumors with scalpels into fragments small enough to be aspirated into a 5 ml pipette without getting stuck at RT. #####%%%%% output "tumor tissue suspension" are used in line10
{"action": "mince", "output": "tumor tissue suspension", "reagent": ["tumors"], "container": ["a 5 ml pipette"]}
Transfer tumor tissue suspension into 50 ml non-vented tissue culture flasks.
{"action": "transfer", "output": "", "reagent": ["tumor tissue suspension"], "container": ["50 ml non-vented tissue culture flasks"]}
Rinse Petri dish with up to 40 ml HBSS .
{"action": "rinse", "output": "", "volume": ["up to 40 ml"]}
transfer the suspension into the flask
{"action": "transfer", "output": "", "container": ["the suspension"]}
Add 5 ml 10x Triple Enzyme Mix to the flask, .
{"action": "add", "output": "", "volume": ["5 ml"], "container": ["the flask"]}
shake the flask at 80 rpm at RT for 1-3 h on a shaker
{"action": "shake", "output": "", "temperature": ["RT"], "device": ["shaker"], "speed": ["80 rpm"], "temperature": ["RT"], "time": ["1-3 h"]}
Repeatedly pipet the tumor cell suspension to further dissociate cells, centrifuge the cell suspension at 50 x g at RT for 10 min . #####%%%%% output "cells" are used in line34
{"action": "centrifuge", "output": "cells", "temperature": ["RT"], "time": ["10 min"], "force": ["50 x g"]}
collect the supernatant #####%%%%% output "the supernatant" are used in line46
{"action": "collect", "output": "the supernatant", "container": [""]}
Discard the bigger pellets in the bottom of the tube .
{"action": "discard", "output": "", "container": ["the tube"]}
centrifuge the supernatant at 200 x g for 5 min #####%%%%% output "the supernatant" are used in line46
{"action": "centrifuge", "output": "the supernatant", "force": ["200 x g"], "time": ["5 min"]}
Suspend the cell pellet with 2 ml ACK lysing buffer for 1 min to deplete red blood cells (observe the cell suspension under a microscope to determine whether red blood cells are depleted completely.
{"action": "suspend", "output": "", "reagent": ["cell pellet"], "volume": ["2 ml"], "time": ["1 min"]}
If there are still too many red blood cells, add more ACK lysing buffer until there are very few red blood cells). #####%%%%% output "cells" are used in line32
{"action": "add", "output": "cells", "reagent": ["more ACK lysing buffer"]}
Re-suspend cells in 15 ml 40% Percoll .
{"action": "re-suspend", "output": "", "reagent": ["cells"], "volume": ["15 ml"]}
add into a 50 ml tube #####%%%%% output "wash buffer" are used in line38
{"action": "add", "output": "wash buffer", "container": ["a 50 ml tube"], "reagent": ["cells"]}
Collect the cells at the interface between 40% Percoll, which should mostly be leukocytes .
{"action": "collect", "output": ""}
Wash the strainer with wash buffer .
{"action": "wash", "output": "", "reagent": ["wash buffer"]}
centrifuge the cell suspension at 425 x g at RT for 10 min #####%%%%% output "tumor" are used in line42
{"action": "centrifuge", "output": "tumor", "temperature": ["RT"], "time": ["10 min"], "force": ["425 x g"]}
Note: After centrifugation, approximately 1.5-2.0 x 105 leukocytes could be obtained from a total of 1 x 106 cells from tumor depending on the tumor type.
{"action": "note", "output": "", "device": ["centrifugation"], "reagent": ["tumor"]}
Re-suspend the cells in desired buffer for further experiments, such as phenotyping with flow cytometry .
{"action": "re-suspend", "output": "", "reagent": ["cells"], "device": ["flow cytometry"]}
Left tube shows that before centrifugation, 80% Percoll is laid under the total cells suspended in 40% Percoll.
{"action": "see", "output": ""}
Right tube shows that after centrifugation, leukocytes are located at the interface between .
{"action": "see", "output": ""}