34
General Methods
Procedure
1. Suspend fragments of tissues to be digested in the trypsin - EDTA solution.
2. Keep this mixture at 20°-30°C. It is not necessary to keep it at 3]oC
because invertebrates are poikilothermic. In general, treatment at lower
temperatures has fewer detrimental effects on cells. During the incubation period, stir the solution occasionally.
3. After an appropriate incubation period (the period should be predetermined), discard the trypsin-EDTA solution, or, if you are treating
cultured cells, add approximately the same volume of culture medium
and suspend the cells by pipetting, and then proceed to step 6.
4. Suspend the tissue fragments in a small amount of culture medium,
and apply vigorous pipetting in order to dissociate cells in the tissue
fragments.
5. Pass the resulting suspension of cells and tissue fragments through a
stainless steel mesh to remove non-dissociated cell masses.
6. Centrifuge the cell suspension at 150 g for 5 min.
7. Discard the supernatant and suspend the cell pellet in fresh medium.
Results
Using this treatment protocol, tissues will be disintegrated, and the result will be the production of a single cell suspension. However, small
cell clumps may be present in the free cell suspension, even after passage
through a stainless steel mesh. In addition, single cells once separated
may form an aggregate again.
Subprotocol 2
Pancreatin Treatment for Dissociation of Cell Masses
Pancreatin is mostly used to detach cultured cells from the substrate.
Materials
Equipment The equipment is the same as that given for subprotocoll.
Solutions - Pancreatin solution
Dissolve 1.12 g NaCl, 0.02 g KCl, 0.1 g glucose, 0.035 g NaHC0 3 , 0.02 g
EDTA, and 0.03125 g pancreatin (Sigma) in approximately 90 ml distilled water. After complete dissolution of the added chemicals, make
the volume up to 100 ml with distilled water. Adjust the pH to 7.2 with
NaOH, and keep the solution frozen at -30°C. This is a pancreatin stock
solution.
General Methods
Procedure
1. Suspend fragments of tissues to be digested in the trypsin - EDTA solution.
2. Keep this mixture at 20°-30°C. It is not necessary to keep it at 3]oC
because invertebrates are poikilothermic. In general, treatment at lower
temperatures has fewer detrimental effects on cells. During the incubation period, stir the solution occasionally.
3. After an appropriate incubation period (the period should be predetermined), discard the trypsin-EDTA solution, or, if you are treating
cultured cells, add approximately the same volume of culture medium
and suspend the cells by pipetting, and then proceed to step 6.
4. Suspend the tissue fragments in a small amount of culture medium,
and apply vigorous pipetting in order to dissociate cells in the tissue
fragments.
5. Pass the resulting suspension of cells and tissue fragments through a
stainless steel mesh to remove non-dissociated cell masses.
6. Centrifuge the cell suspension at 150 g for 5 min.
7. Discard the supernatant and suspend the cell pellet in fresh medium.
Results
Using this treatment protocol, tissues will be disintegrated, and the result will be the production of a single cell suspension. However, small
cell clumps may be present in the free cell suspension, even after passage
through a stainless steel mesh. In addition, single cells once separated
may form an aggregate again.
Subprotocol 2
Pancreatin Treatment for Dissociation of Cell Masses
Pancreatin is mostly used to detach cultured cells from the substrate.
Materials
Equipment The equipment is the same as that given for subprotocoll.
Solutions - Pancreatin solution
Dissolve 1.12 g NaCl, 0.02 g KCl, 0.1 g glucose, 0.035 g NaHC0 3 , 0.02 g
EDTA, and 0.03125 g pancreatin (Sigma) in approximately 90 ml distilled water. After complete dissolution of the added chemicals, make
the volume up to 100 ml with distilled water. Adjust the pH to 7.2 with
NaOH, and keep the solution frozen at -30°C. This is a pancreatin stock
solution.