The following protocol describes the use of trypsin to treat cells for passaging or collection. Cell lines are usually maintained by passaging once a week and changing the culture medium the day before passaging; however, some cell lines require more frequent passaging. Each cell line should be passaged separately to avoid cross contamination of the cell lines. No more than one cell line can be placed in the ultra-clean bench at the same time. This experiment is derived from the Cell Lab Guide (previous volume) by Huang Peitang.
Operation method
Trypsin treatment of monolayer cells
Principle
The following protocol describes the use of trypsin to treat cells for passaging or collection. Cell lines are usually maintained by passaging once a week and changing the culture medium the day before passaging; however, some cell lines require more frequent passaging. Each cell line should be passaged separately to avoid cross contamination of the cell lines. No more than one cell line can be placed in the ultra-clean bench at the same time. Move 1) Aspirate the culture solution from the cell culture flask or petri dish in which the monolayer of cells was cultured. Caveat Caution.- The amount of trypsin in this passaging protocol may be reduced if a solution containing EDTA chelating agent is used as the prewash solution. In such a case, Step 2 may be modified by using a trypsin-free prewash solution.Pre-wash solutionEDTA. tetrasodium salt 2.0 gNaCl 8.0 gKCl 0.4 gGlucose 1.0 gNaHCO30.35 g1% phenol red 1 mlH2O 900 mlAdjust pH to 7.2- For cell lines that are not very sensitive to trypsin, the following rapid trypsinization protocols are availablea. Wash monolayer of cells with PBS.b. Add enough lx trypsin working solution to cover all cells and incubate at 37°C for 5 minutes. c. Check for cell detachment and incubate at 37°C for 5 minutes.c. Check for cell detachment, aspirate the cells from the trypsin working solution and dilute with culture medium to the number of cells to be inoculated. For more product details, please visit Aladdin Scientific website.
2) Add 0.05% trypsin working solution (1 ml for T-25 cell culture flask; 3 ml for T-75 cell culture flask).
Sterile 10x or 1x Trypsin/EDTA Working Solution can be purchased from Life Technologies or prepared according to the following recipe:
Trypsin 1x Working Solution (0.05%)
Trypsin/EDTA 10x stock solution 100 ml
NaCl 7.1g
KCl 0.4 g
Glucose 1.0 g
NaHCO3 0.35 g
1% Phenol Red 1.0ml
H2O 900 ml
Adjust pH to 7.2
Caution: KCl, phenol red
Trypsin/EDTA 10X stock solution
Trypsin (1:250) 5.0 g
EDTA-tetrasodium salt 2.0 g
NaCl 0.85 g
H2O 100 ml
-20°C Store Trypsin/EDTA 10x stock solution and 1x working solution. Smaller fractions can be stored at 4°C for 1-2 weeks. Trypsin should be stored at 37°C for as short a time as possible to avoid degradation of trypsin and loss of enzyme activity.
3) Rapidly rotate the cell culture flasks back and forth 4-5 times so that trypsin coats all cells in the flasks and traces of culture medium and serum are diluted.
4) Check the cells, do not wait until the cells are detached before aspirating the trypsin solution.
5) Add another 1-3 ml of trypsin working solution and rotate the flask 4-5 times.
6) Be careful to aspirate the trypsin solution before the cells are detached.
7) Loosely screw the cap on the cell culture flask and place the flask in the incubator at 37°C for 2~5 minutes.
8) Tap the cell culture flask with your hand so that a small amount of liquid in the flask can bring down the cells that have to be detached, and check the cell detachment situation. If no cells are detached, place the flask back in the incubator at 37°C for a few minutes. Continue tapping the flask until the cells are completely detached.
9) Resuspend the cells in cell culture medium (2-5 ml/T-25 cell culture flask).
10) Blow the cells gently to disperse the agglomerates and inoculate the cells at the desired cell density.