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Methods for Setting Up Primary Cultures Specific to Animal Groups
6. Remove 12-13-day-old eggs. The stage of eggs used is important.
Dissociation 1. Place the eggs in a sterile glass petri dish (see Comments).
2. Add a few drops of culture medium, just enough to cover the eggs but
not enough to allow them to float.
3. Crush the eggs with a glass rod.
4. Filter the resulting tissue and egg shell suspension through a sterile
coarse sintered glass funnel (pore diameter 100-200 flm).
Culture 1. Centrifuge the filtrate at 280 g for 10 min.
set-up 2. Discard the supernatant and suspend the cell pellet in PBSA.
3. Centrifuge the cell suspension at 280 g for 10 min.
4. Repeat the steps 2 and 3.
5. Discard the supernatant and suspend the cell pellet in Leibovitz's LIS medium.
6. Transfer the cell suspension into a culture vessel.
7. Culture at 28°C (see Comments).
8. Replace half the medium once a week.
Troubleshooting
If an incomplete monolayer is formed, reseed the cells as follows:
1. Replace half the medium with fresh medium.
2. Remove the cells from the substrate by means of a rubber policeman.
3. Pipette the cells gently and allow them to reattach in the original vessel.
Comments
In order to obtain an adequate seeding cell density when first setting up
the culture, make up a 4-ml culture with the eggs of 10 female ticks.
Attachment of the cells to the substrate is very important for later cell
growth. Therefore, disturbance of the cells, such as when moving the
culture vessel, should be minimized. Cell attachment may take several
days.
Trypsin is detrimental to cells and should not be used for detaching
cells at subculture.
Perform early subcultures by using a 1 : 1 mixture of conditioned medium and fresh medium with a high cell density. For this, cells from two
vessels may be pooled and seeded into one vessel.
Results
After approximately 1 week, fibroblast-type cells and granular cells appear between the attached cell clumps (Fig. O. By approximately four
weeks, small round cells replace these fibroblast-type and granular cells.
When the small round cell type multiplies to cover the bottom of the
Methods for Setting Up Primary Cultures Specific to Animal Groups
6. Remove 12-13-day-old eggs. The stage of eggs used is important.
Dissociation 1. Place the eggs in a sterile glass petri dish (see Comments).
2. Add a few drops of culture medium, just enough to cover the eggs but
not enough to allow them to float.
3. Crush the eggs with a glass rod.
4. Filter the resulting tissue and egg shell suspension through a sterile
coarse sintered glass funnel (pore diameter 100-200 flm).
Culture 1. Centrifuge the filtrate at 280 g for 10 min.
set-up 2. Discard the supernatant and suspend the cell pellet in PBSA.
3. Centrifuge the cell suspension at 280 g for 10 min.
4. Repeat the steps 2 and 3.
5. Discard the supernatant and suspend the cell pellet in Leibovitz's LIS medium.
6. Transfer the cell suspension into a culture vessel.
7. Culture at 28°C (see Comments).
8. Replace half the medium once a week.
Troubleshooting
If an incomplete monolayer is formed, reseed the cells as follows:
1. Replace half the medium with fresh medium.
2. Remove the cells from the substrate by means of a rubber policeman.
3. Pipette the cells gently and allow them to reattach in the original vessel.
Comments
In order to obtain an adequate seeding cell density when first setting up
the culture, make up a 4-ml culture with the eggs of 10 female ticks.
Attachment of the cells to the substrate is very important for later cell
growth. Therefore, disturbance of the cells, such as when moving the
culture vessel, should be minimized. Cell attachment may take several
days.
Trypsin is detrimental to cells and should not be used for detaching
cells at subculture.
Perform early subcultures by using a 1 : 1 mixture of conditioned medium and fresh medium with a high cell density. For this, cells from two
vessels may be pooled and seeded into one vessel.
Results
After approximately 1 week, fibroblast-type cells and granular cells appear between the attached cell clumps (Fig. O. By approximately four
weeks, small round cells replace these fibroblast-type and granular cells.
When the small round cell type multiplies to cover the bottom of the