Dilute blood 1:1 in PBS, add 15 ml histopaque-1077 to a 50 ml conical tube.
{"action": "dilute", "output": "", "reagent": ["blood", "PBS"]}
Collect WBC at interphase using a Pasteur pipette, avoid aspirating histopaque-1077. #####%%%%% output "interphase" are not used
{"action": "collect", "output": "interphase", "reagent": ["WBC"], "device": ["Pasteur pipette"]}
Pellet cells at 300 x g for 10 min (RT), wash in cold PBS at 300 x g for 10 min, .
{"action": "pellet", "output": "", "reagent": ["cells"], "speed": ["300 x g"], "time": ["10 min"], "temperature": ["RT"]}
add 50 ml PBS
{"action": "add", "output": "", "volume": ["50 ml"], "reagent": ["PBS"]}
take a sample for pre-sort control for FACs analysis #####%%%%% output "FACs analysis" are not used
{"action": "take", "output": "FACs analysis", "volume": [""]}
Centrifuge at 300 x g for 10 min (RT), re-, add 100 µl FcR Blocking reagent Microbead dendritic antibody cocktail.
{"action": "centrifuge", "output": "", "speed": ["300 x g"], "time": ["10 min"], "temperature": ["RT"]}
suspend pellet in 300 µl MACS buffer per 10^8 cells
{"action": "suspend", "output": "", "reagent": ["pellet"], "volume": ["300 µl"]}
Incubate for 10 min at 4 °C, spin at 300 x g for 10 min (RT).
{"action": "incubate", "output": "", "temperature": ["4 \u00b0C"], "time": ["10 min (RT)"]}
Charge the LS column with 3 ml MACS buffer, discard flow-through. #####%%%%% output "flow-through" are not used
{"action": "charge", "output": "", "device": ["LS column"], "reagent": ["MACS buffer"], "volume": ["3 ml"]}
Re-suspend cells in 500 µl MACS buffer per 10^8 cells, add to column, . #####%%%%% output "column" are not used
{"action": "re-suspend", "output": "", "reagent": ["cells"], "volume": ["500 µl"]}
Count cells, use for downstream applications.
{"action": "count", "output": "", "reagent": ["cells"]}
For FACS staining, pellet 10^5 cells at 400 x g for 5 min, -, add 1 µl normal mouse serum, .
{"action": "pellet", "output": "", "reagent": ["cells"], "speed": ["400 x g"], "time": ["5 min"]}
incubate for 10 min (RT) #####%%%%% output "directly conjugated" are used in line28
{"action": "incubate", "output": "directly conjugated", "time": ["10 min"], "temperature": ["RT"]}
Add directly conjugated antibody, spin at 400 x g for 5 min (RT), -, .
{"action": "add", "output": "", "reagent": ["directly conjugated antibody"]}
suspend in 500 µl
{"action": "suspend", "output": "", "volume": ["500 µl"]}
proceed to FACS analysis
{"action": "proceed", "output": "", "device": ["FACS analysis"]}