Rinse MSC with 2 ml PBS, .
{"action": "rinse", "output": "", "reagent": ["MSC"], "volume": ["2 ml PBS"]}
trypsinize with 0.5 ml trypsin,
{"action": "trypsinize", "output": "", "volume": ["0.5 ml trypsin"]}
transfer to 15 ml tube, #####%%%%% output "FBS" are used in line8
{"action": "transfer", "output": "FBS", "container": ["15 ml tube"]}
add 10 ml DMEM with 10% FBS
{"action": "add", "output": "", "volume": ["10 ml DMEM"], "reagent": ["FBS"]}
Incubate overnight at 37 °C, 5% CO2. #####%%%%% output "osteogenic differentiation medium" are used in line12
{"action": "incubate", "output": "osteogenic differentiation medium", "temperature": ["37 \u00b0C, 5% CO2"]}
Wash cells twice with 2 ml PBS, add osteogenic differentiation medium, .
{"action": "wash", "output": "", "volume": ["2 ml PBS"], "reagent": ["osteogenic differentiation medium"]}
change medium every 2 days for 10 days #####%%%%% output "Alizarin red S" are used in line16
{"action": "change", "output": "Alizarin red S", "time": ["every 2 days for 10 days"]}
At day 10, wash cells with PBS, stain with Alizarin red S for 5 min, . #####%%%%% output "100 μl urea/phosphate lysis buffer" are used in line18
{"action": "rinse", "output": "100 \u03bcl urea/phosphate lysis buffer", "reagent": ["PBS", "Alizarin red S"], "time": ["5 min"]}
Wash cells 5 times with 1 ml ice-cold serum-free DMEM, scrape in 100 μl urea/phosphate lysis buffer. #####%%%%% output "SDS-PAGE loading buffer" are used in line20
{"action": "scrape", "output": "SDS-PAGE loading buffer", "reagent": ["100 \u03bcl urea/phosphate lysis buffer"]}
Transfer lysates to 1.5 ml tubes, dilute 10 times with SDS-PAGE loading buffer, .
{"action": "dilute", "output": "", "concentration": ["10 times"], "reagent": ["SDS-PAGE loading buffer"]}
quantify protein concentration,
{"action": "quantify", "output": "", "concentration": ["protein concentration"]}
analyze 50 μg protein by SDS-PAGE blot #####%%%%% output "SDS-PAGE blot" are not used
{"action": "evaluate", "output": "SDS-PAGE blot", "reagent": [""]}
Detect IL-27 using biotinylated goat anti-mouse p28 Ab .
{"action": "detect", "output": "", "reagent": ["IL-27", "biotinylated goat anti-mouse p28 Ab"]}