Passaging adherent cells
#3_cell
Aim
Passage adherent cells to a new dish for maintenance of the cell culture.
Materials
General notes:
All solutions and consumables should be sterile
Clean the clean bench with 70% Ethanol before and after use
PBS, stored at RT
0.25% Trypsin, 0.02% EDTA, PBS stored at 4℃
PBS 89 mL
2.5% Trypsin 10 mL
2% EDTA 1 mL
Total 100 mL
Culture medium (e.g. DMEM high glucose, 10% FBS), stored at 4℃
DMEM high glucose (Nacalai) 500 mL
Heat inactivated (56℃, 30 min) FBS (Sigma) 50 mL
Plastic 10 cm dish
Methods
Prepare culture medium and trypsin solution.
Check the cells in the microscope and decide how dense to passage the cells, e.g. 1:5 or 1:10.
Aspirate the supernatant medium from the 10 cm dish.
Add 1 mL PBS, distribute over all cells, and aspirate.
Add 1 mL Trypsin solution, distribute over all cells, and aspirate.
Incubate the cells at 37℃ for 5 min
Check whether cells are detached, by eye or in the microscope.
Add fresh culture medium to a new 10 cm dish, e.g. 9 mL for 1:10 passage or 8 mL for 1:5 passage.
Add 10 mL culture medium to the 10 cm dish with detached cells and suspend cells in the medium by pipetting up and down, and add 1 mL for 1:10 passage or 2 mL for 1:5 passage to the new 10 cm dish.
Rock the plate forward/backwards 10 times and left/right 10 times (don’t swirl, otherwise cells might gather in the middle of the dish).
Incubate the cells at 37℃, 5% CO2.
Remarks
Date : 2024/12/25
Modified Date :
Author : y-goto.icon