Sack: Cell Splitting Protocol: Difference between revisions
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Notes: Steps should be done inside of a Biological Safety Cabinet in Sterile conditions. Make sure that the area has been cleaned with 70% ethanol, the UV light is off, and that the blower has been on for at least 10 min before starting. Once finished close clean the area off with 70% ethanol, close the cabinet and turn on the UV light. | Notes: Steps should be done inside of a Biological Safety Cabinet in Sterile conditions. Make sure that the area has been cleaned with 70% ethanol, the UV light is off, and that the blower has been on for at least 10 min before starting. Once finished close clean the area off with 70% ethanol, close the cabinet and turn on the UV light. | ||
Procedures (done in T25 flask):<BR> | Procedures (done in T25 flask):<BR> | ||
# Check under microscope to make sure that cells are less than 80% confluent<BR> | # Check under microscope to make sure that cells are less than 80% confluent<BR> | ||
# Remove the solution from the flask<BR> | # Remove the solution from the flask<BR> | ||
# Rinse with DPBS (Divalent free Dulbecco’s Phosphate Buffered Saline) ~ 5 mL<BR> | # Rinse with DPBS (Divalent free Dulbecco’s Phosphate Buffered Saline) ~ 5 mL<BR> | ||
# Remove the solution<BR> | |||
# Remove the solution<BR> | |||
# Add 1 mL 0.05% Trypsin EDTA<BR> | # Add 1 mL 0.05% Trypsin EDTA<BR> | ||
# Place in 37° C incubator until cells detach from the dish ~ 5 min<BR> | # Place in 37° C incubator until cells detach from the dish ~ 5 min<BR> | ||
#*Check under the microscope to make sure cells have detached<BR> | #*Check under the microscope to make sure cells have detached<BR> | ||
# Add 5 mL of Cell Media into a new dishes<BR> | # Add 5 mL of Cell Media into a new dishes<BR> | ||
# Check under the microscope to make sure the cells are dispersed into single cells.<BR> | |||
# | |||
# Add the appropriate amount of cells into the new dishes<BR> | # Add the appropriate amount of cells into the new dishes<BR> | ||
# Swirl the dish or pipet up and down<BR> | # Swirl the dish or pipet up and down<BR> | ||
# Label the Dish: Name, Date, Cell Line, Passage #, Ratio<BR> | # Label the Dish: Name, Date, Cell Line, Passage #, Ratio<BR> | ||
*Cell Media: Ham’s – F12 from Gibco, 10% FBS, Penicillin/Streptomycin | *Cell Media: Ham’s – F12 from Gibco, 10% FBS, Penicillin/Streptomycin | ||
To make add | To make add 500 mL Media + 50 mL FBS |
Latest revision as of 12:20, 10 August 2013
Notes: Steps should be done inside of a Biological Safety Cabinet in Sterile conditions. Make sure that the area has been cleaned with 70% ethanol, the UV light is off, and that the blower has been on for at least 10 min before starting. Once finished close clean the area off with 70% ethanol, close the cabinet and turn on the UV light. Procedures (done in T25 flask):
To make add 500 mL Media + 50 mL FBS |