212
Methods for Setting Up Primary Cultures Specific to Animal Groups
Dissociation
Separation of
cell types
Cell fusion
Culture
set-up
Selection of
hybridoma
Continuous
culture
Procedure
1. Collect the metacestode tissues from jird under sterile conditions.
2. Mince the tissues using a surgical scalpel.
3. Wash the tissues thoroughly with cold IBE to remove any contaminating host blood cells.
4. Digest the tissues in collagenase solution at 37°C for 60 min.
5. Remove undigested larger tissue fragments by passing the solution
through a 380-llm nylon sieve.
6. Separate proto scoleces from the remaining digested tissues by further passage through a 50-11m nylon sieve.
7. Leave the remaining suspension, containing liberated single cells and
calcareous corpuscles, to stand for 10 min to allow sedimentation of
the latter cell type.
8. Centrifuge the supernatant at 400 g for 5 min.
9. Wash the pellet twice in cold IBE.
1. Apply discontinuous Percoll gradient (consisting of an equal volume
of 100 %,50 %,40 % and 30 % isotonic Percoll, diluted to the appropriate concentration with lEE) to the washed cells for the separation
of the different types of cells. Do not overlay the gradient with more
than 2 X 10 7 cells/ml gradient.
2. Centrifuge the gradient at 1,000 g at room temperature for 40 min.
This gradient centrifugation results in the separation of four bands.
3. Take the second band from the top (density 1.056 g/ml), which contains exclusively Echinococcus germinal cells.
1. Mix 3 x 10 7 germinal cells and the same number of mouse myeloma
cells.
2. Centrifuge at 600 g for 7 min.
3. Discard the supernatant.
4. Disrupt the cell pellet by gently tapping the bottom of the tube.
5. Add 1.0 ml of 50% PEG 4000 in DMEM to the cell pellet drop-wise
over a period of 60 s.
6. Keep the resulting cell suspension at 37°C for 1 min.
7. Add 1 ml DMEM drop-wise over a period of 60 s.
8. Add a further 20 ml DMEM over a period of 5 min.
1. Centrifuge the cells at 600 g for 5 min.
2. Gently suspend the cells in DMEM containing 20% FBS.
3. Distribute the cell suspension in 2-ml wells in Linbro BCL-S041 trays.
1. Replace half the medium with HAT medium after 24 h.
2. Repeat step lover the next 2 days, and then every 2 days.
1. When vigorous growth is obtained, transfer the cells into aminopterinfree HAT medium for 4-7 days, and thereafter into DMEM.
Methods for Setting Up Primary Cultures Specific to Animal Groups
Dissociation
Separation of
cell types
Cell fusion
Culture
set-up
Selection of
hybridoma
Continuous
culture
Procedure
1. Collect the metacestode tissues from jird under sterile conditions.
2. Mince the tissues using a surgical scalpel.
3. Wash the tissues thoroughly with cold IBE to remove any contaminating host blood cells.
4. Digest the tissues in collagenase solution at 37°C for 60 min.
5. Remove undigested larger tissue fragments by passing the solution
through a 380-llm nylon sieve.
6. Separate proto scoleces from the remaining digested tissues by further passage through a 50-11m nylon sieve.
7. Leave the remaining suspension, containing liberated single cells and
calcareous corpuscles, to stand for 10 min to allow sedimentation of
the latter cell type.
8. Centrifuge the supernatant at 400 g for 5 min.
9. Wash the pellet twice in cold IBE.
1. Apply discontinuous Percoll gradient (consisting of an equal volume
of 100 %,50 %,40 % and 30 % isotonic Percoll, diluted to the appropriate concentration with lEE) to the washed cells for the separation
of the different types of cells. Do not overlay the gradient with more
than 2 X 10 7 cells/ml gradient.
2. Centrifuge the gradient at 1,000 g at room temperature for 40 min.
This gradient centrifugation results in the separation of four bands.
3. Take the second band from the top (density 1.056 g/ml), which contains exclusively Echinococcus germinal cells.
1. Mix 3 x 10 7 germinal cells and the same number of mouse myeloma
cells.
2. Centrifuge at 600 g for 7 min.
3. Discard the supernatant.
4. Disrupt the cell pellet by gently tapping the bottom of the tube.
5. Add 1.0 ml of 50% PEG 4000 in DMEM to the cell pellet drop-wise
over a period of 60 s.
6. Keep the resulting cell suspension at 37°C for 1 min.
7. Add 1 ml DMEM drop-wise over a period of 60 s.
8. Add a further 20 ml DMEM over a period of 5 min.
1. Centrifuge the cells at 600 g for 5 min.
2. Gently suspend the cells in DMEM containing 20% FBS.
3. Distribute the cell suspension in 2-ml wells in Linbro BCL-S041 trays.
1. Replace half the medium with HAT medium after 24 h.
2. Repeat step lover the next 2 days, and then every 2 days.
1. When vigorous growth is obtained, transfer the cells into aminopterinfree HAT medium for 4-7 days, and thereafter into DMEM.