84
Methods for Setting Up Primary Cultures Specific to Animal Groups
3. To form a high-density cushion during cell separation, supplement
the basic medium with 20% Ficoll.
4. Sterilize all media by filtration through a 0.2-llm Nalgen filter, and
store at 4°C until use.
5. Fractionation of cells is performed using a Celsep apparatus. First, fill
the Celsep chamber (a flat cylindrical chamber with a height of 25
mm; 11 capacity), positioned at a 30° angle with the basic medium
containing a continuous gradient of 1.5%-3.5% Ficoll from the bottom opening of the chamber. The gradient can be formed by a gradient maker, and is delivered by a peristaltic pump at 30 ml/min.
6. Deliver approximately 100 ml cushion medium into the chamber after the gradient from the bottom opening.
7. Introduce 30 ml cell suspension containing 3 x 10 7 cells onto the gradient using the peristaltic pump at a rate of 10 ml/min from the top
opening of the chamber.
8. Deliver approximately 50 ml of the overlay medium onto the cell suspension in the same manner as above.
9. Pack the apparatus in ice, and leave for 2.5 h in a horizontal position
to allow cells to sediment.
10. Reorientate the Celsep chamber to a 30° angle, and collect 20 fractions, each containing 50 ml medium, into sterile centrifuge tubes.
11. Centrifuge for 10 min at 350 g to collect cells.
12. Wash the cells once in Schneider's medium containing 20% FBS.
13. Resuspend the cells in the above medium, and transfer to a culture
vessel.
14. Culture at 25°C.
Results
The larger and denser cells in the first fraction will differentiate to yield
pure neuronal cultures, as judged by morphologic, immunologic, and
biochemical criteria. Cells in the last fraction will differentiate into a predominantly muscle-enriched cell population. During culture, neuronal
cells divide to some extent. This method is highly reproducible.
Subprotocol 6
Hemocytes
So far, only hemocytes of two recessive-lethal mutants of D. melanogaster
have been cultured. This mutants are characterized by malignant blood
neoplasms. Following is the method to culture these neoplastic hemocytes
developed by Gateff et al. (1980).
Methods for Setting Up Primary Cultures Specific to Animal Groups
3. To form a high-density cushion during cell separation, supplement
the basic medium with 20% Ficoll.
4. Sterilize all media by filtration through a 0.2-llm Nalgen filter, and
store at 4°C until use.
5. Fractionation of cells is performed using a Celsep apparatus. First, fill
the Celsep chamber (a flat cylindrical chamber with a height of 25
mm; 11 capacity), positioned at a 30° angle with the basic medium
containing a continuous gradient of 1.5%-3.5% Ficoll from the bottom opening of the chamber. The gradient can be formed by a gradient maker, and is delivered by a peristaltic pump at 30 ml/min.
6. Deliver approximately 100 ml cushion medium into the chamber after the gradient from the bottom opening.
7. Introduce 30 ml cell suspension containing 3 x 10 7 cells onto the gradient using the peristaltic pump at a rate of 10 ml/min from the top
opening of the chamber.
8. Deliver approximately 50 ml of the overlay medium onto the cell suspension in the same manner as above.
9. Pack the apparatus in ice, and leave for 2.5 h in a horizontal position
to allow cells to sediment.
10. Reorientate the Celsep chamber to a 30° angle, and collect 20 fractions, each containing 50 ml medium, into sterile centrifuge tubes.
11. Centrifuge for 10 min at 350 g to collect cells.
12. Wash the cells once in Schneider's medium containing 20% FBS.
13. Resuspend the cells in the above medium, and transfer to a culture
vessel.
14. Culture at 25°C.
Results
The larger and denser cells in the first fraction will differentiate to yield
pure neuronal cultures, as judged by morphologic, immunologic, and
biochemical criteria. Cells in the last fraction will differentiate into a predominantly muscle-enriched cell population. During culture, neuronal
cells divide to some extent. This method is highly reproducible.
Subprotocol 6
Hemocytes
So far, only hemocytes of two recessive-lethal mutants of D. melanogaster
have been cultured. This mutants are characterized by malignant blood
neoplasms. Following is the method to culture these neoplastic hemocytes
developed by Gateff et al. (1980).