4 General Cell Culture Methods
35
Add 85 ml distilled water to 10 ml pancreatin stock solution, and dissolve 1.12 gNaCI,0.02 g KCI,O.1 g glucose, 0.035 gNaHC0 3 ,and 0.02 g
EDTA in this mixture. Add distilled water to the solution to make the
volume up to 100 ml.Adjust the pH of the solution to 7.2 with NaOH.
This is a ready-to-use pancreatin solution.
Procedure
1. Remove the culture medium from a monolayer culture.
2. Add the pancreatin solution so as to cover the whole cell sheet.
3. Incubate at 25°C for 5-10 min. The duration of treatment varies depending on the cell line used and should be determined by preliminary experiments.
4. When most cells become ready to detach, add an appropriate amount
of culture medium.
5. Detach the cells by pipetting.
6. Follow steps 6 and 7 of the procedure given in subprotocol1.
Results
The results are the same as those observed following treatment with
trypsin.
Subprotocol 3
Dispase Treatment for Dissociation of Cell Masses
Dispase is a protease produced by Bacillus polymixa. A commercialized
product is available. The optimum pH is 7.5-8.5, but dispase is stable
over the pH range 5-9.
Materials
The equipment used is the same as that given for subprotocol1.
Equipment
- Dispase solution
Solutions
Dissolve 5 g dispase (Godo Shusei) in 100 ml distilled water.
This results in a dark brown solution. The filter-sterilized solution
can be stored below -20°C over a long period of time.
Procedure
1. Suspend the tissue fragments to be digested in culture medium.
2. Add the dispase solution at 1/10 the volume of the medium added to
35
Add 85 ml distilled water to 10 ml pancreatin stock solution, and dissolve 1.12 gNaCI,0.02 g KCI,O.1 g glucose, 0.035 gNaHC0 3 ,and 0.02 g
EDTA in this mixture. Add distilled water to the solution to make the
volume up to 100 ml.Adjust the pH of the solution to 7.2 with NaOH.
This is a ready-to-use pancreatin solution.
Procedure
1. Remove the culture medium from a monolayer culture.
2. Add the pancreatin solution so as to cover the whole cell sheet.
3. Incubate at 25°C for 5-10 min. The duration of treatment varies depending on the cell line used and should be determined by preliminary experiments.
4. When most cells become ready to detach, add an appropriate amount
of culture medium.
5. Detach the cells by pipetting.
6. Follow steps 6 and 7 of the procedure given in subprotocol1.
Results
The results are the same as those observed following treatment with
trypsin.
Subprotocol 3
Dispase Treatment for Dissociation of Cell Masses
Dispase is a protease produced by Bacillus polymixa. A commercialized
product is available. The optimum pH is 7.5-8.5, but dispase is stable
over the pH range 5-9.
Materials
The equipment used is the same as that given for subprotocol1.
Equipment
- Dispase solution
Solutions
Dissolve 5 g dispase (Godo Shusei) in 100 ml distilled water.
This results in a dark brown solution. The filter-sterilized solution
can be stored below -20°C over a long period of time.
Procedure
1. Suspend the tissue fragments to be digested in culture medium.
2. Add the dispase solution at 1/10 the volume of the medium added to