Preparations
- Medium (e.g., DMEM, 10% FBS)
- PET (PBS/0.01% EDTA/0.25% Trypsin) solution
- (For cells with strong cell-to-cell contact such as MDCK cells) PBS/1 mM EDTA
Passage procedures for cultured cells (like HeLa and 293T cells)
- (Important) Before starting the following steps, you must check cell condition by microscope, such as confluency, morphology, etc.
- Warm medium and PET solution to 37 degrees.
- Move medium, PET solution and culture cell dish into safety cabinet.
- Aspirate the medium in the culture dish.
- Add 1 mL PET solution to the dish, spread the PET solution throughout the surface of the dish.
- This is the washing process to remove serum, which contains trypsin inhibitor.
- Aspirate the PET solution.
- Add 1 mL PET solution to the dish, spread the PET solution throughout the surface of the dish.
- Aspirate the PET solution, and incubate the dish for 4-5 min in a humidified CO2 incubator.
- The tiny amount of PET solution covering the surface on culture dish is enough to detach cells from the dish.
- This process also reduces trypsin carry-over.
- (Important) Check whether cells are really detached from the dish under a microscope.
- Suspend the cells with the serum-containing medium by pipetting with 10 mL disposable pipet.
- Residual trypsin must be inhibited by serum.
- Split the suspended cells into new culture dishes with the new medium.
- HeLa cells: 1:5 and 1:10 ratio for 2 and 3 days culture, respectively.
Passage procedures for epithelial cells showing strong cell-to-cell contact (like MDCK cells)
- (Important) Before starting the following steps, you must check cell condition by microscope, such as confluency, morphology, etc.
- Warm medium, PET solution and PBS/1 mM EDTA solution to 37 degrees.
- Move medium, PET solution, PBS/1 mM EDTA solution and culture cell dish into safety cabinet.
- Aspirate the medium in the culture dish.
- Add 7-10 mL PBS/1 mM EDTA to the dish, and incubate the dish for 5 min in a humidified CO2 incubator.
- This process allows attenuating cell-to-cell contact of the cells.
- Aspirate the PBS/1 mM EDTA solution in the culture dish.
- Add 1 mL PET solution to the dish, spread the PET solution throughout the surface of the dish.
- Aspirate the PET solution, and incubate the dish for 3-5 min in the humidified CO2 incubator.
- The tiny amount of PET solution covering the surface on culture dish is enough to detach cells from the dish.
- This process also reduces trypsin carry-over.
- (Important) Check whether cells are really detached from the dish under a microscope.
- Suspend the cells with the serum-containing medium by pipetting with 10 mL disposable pipet.
- Residual trypsin must be inhibited by serum.
- Split the suspended cells into new culture dishes with the new medium.
- MDCK cells: 1:6 and 1:12 ratio for 2 and 3 days culture, respectively.
Passage procedures for MCF10A cells
Medium
- DMEM/F12 (phenol-red free): 500 mL
- Horse Serum: 25 mL
- Pen/Strep: 5.0 mL
- EGF (100 ug/mL): 100 μL
- Hydrocortizone (1 mg/mL): 250 μL
- Insulin (10 mg/mL): 500μL
- CholeraToxin (1 mg/mL): 50μL
- (Important) Before starting the following steps, you must check cell condition by microscope, such as confluency, morphology, etc.
- Warm medium, modified PET (PBS/0.01% EDTA/0.025% Trypsin) solution to 37 degrees.
- Move medium, PET solution, PBS/1 mM EDTA solution and culture cell dish into safety cabinet.
- Aspirate the medium in the culture dish.
- Add 7 mL modified PET (PBS/0.01% EDTA/0.025% Trypsin) solution to the dish, and incubate the dish for 15-25 min in a humidified CO2 incubator.
- Suspend the cells by pipetting with 10 mL disposable pipet.
- Transfer the suspended cells to a 15 mL falcon tube.
- Centrifuge (typically 1000 rpm for 3 min).
- Remove the supernatant by aspiration. Do not aspirate the precipitated cells.
- Add 10 mL Medium and resuspend the cells by pipetting.
- Split the resuspended cells into new culture dishes with the new medium.
- MCF10A cells: 1:5 and 1:10 ratio for 3 and 4 days culture, respectively.