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Methods for Setting Up Primary Cultures Specific to Animal Groups
Antibiotics - Streptomycin sulfate
- Penicillin sodium salt
- Nystatin
Animals - Botryllus schlosseri
- Allow the larvae to settle on the settlement glass slide
- Raise the larvae in circulating filtered seawater at 20°C, under aeration through an airstone and 14110 h lightldark regimen
- Change the seawater three times a week
- Clean the animals with soft brushes carefully before blood collection
- Clean the glass slides thoroughly also, and wipe them with 70%
ethanol
- Transfer the slides with the animals to a 11 tank containing seawater passed through a 0.45-llm membrane filter (Millipore), and a
mixture of antibiotics, which contains streptomycin sulfate 10 mgl
ml, penicillin sodium salt 10 4 IU/ml, and nystatin 1,250 IU/ml, at a
concentration of 1 ml/100 ml seawater and leave the animals there
for 48 h
- It is preferable that no food be supplied to the animals during this
period in order to reduce bacterial contamination of the cell culture.
Procedure
Bleeding 1. Select large colonies composed of at least 100 zooids each (cf. Fig. 2).
2. Rinse the colonies several times with WS.
3. Peal the colonies from the glass slides with sterile single-edged surgical razor blades.
4. Place one of the colonies in a petri dishes (9 cm) containing ice-cold
WS.
5. Tease the tunic matrix and zooids apart using forceps.
6. Collect the exuded blood and accumulate it in ice-cold WS.
Culture 1. Remove the tissues and tunic suspended in the WS.
set-up 2. Filter the ws containing blood cells through a 30-llm nylon mesh.
3. Centrifuge the filtrate gently at 300 g for 10 min at 4°C.
4. Resuspend the cell pellet in an ice-cold WS, and centrifuge at 300 g for
10 min at 4°C.
5. Repeat step 4 five times.
6. Suspend the final pellet from step 5 in ice-cold BCCM.
7. Distribute the cell suspension to the wells of 24-well tissue culture
plates.
Methods for Setting Up Primary Cultures Specific to Animal Groups
Antibiotics - Streptomycin sulfate
- Penicillin sodium salt
- Nystatin
Animals - Botryllus schlosseri
- Allow the larvae to settle on the settlement glass slide
- Raise the larvae in circulating filtered seawater at 20°C, under aeration through an airstone and 14110 h lightldark regimen
- Change the seawater three times a week
- Clean the animals with soft brushes carefully before blood collection
- Clean the glass slides thoroughly also, and wipe them with 70%
ethanol
- Transfer the slides with the animals to a 11 tank containing seawater passed through a 0.45-llm membrane filter (Millipore), and a
mixture of antibiotics, which contains streptomycin sulfate 10 mgl
ml, penicillin sodium salt 10 4 IU/ml, and nystatin 1,250 IU/ml, at a
concentration of 1 ml/100 ml seawater and leave the animals there
for 48 h
- It is preferable that no food be supplied to the animals during this
period in order to reduce bacterial contamination of the cell culture.
Procedure
Bleeding 1. Select large colonies composed of at least 100 zooids each (cf. Fig. 2).
2. Rinse the colonies several times with WS.
3. Peal the colonies from the glass slides with sterile single-edged surgical razor blades.
4. Place one of the colonies in a petri dishes (9 cm) containing ice-cold
WS.
5. Tease the tunic matrix and zooids apart using forceps.
6. Collect the exuded blood and accumulate it in ice-cold WS.
Culture 1. Remove the tissues and tunic suspended in the WS.
set-up 2. Filter the ws containing blood cells through a 30-llm nylon mesh.
3. Centrifuge the filtrate gently at 300 g for 10 min at 4°C.
4. Resuspend the cell pellet in an ice-cold WS, and centrifuge at 300 g for
10 min at 4°C.
5. Repeat step 4 five times.
6. Suspend the final pellet from step 5 in ice-cold BCCM.
7. Distribute the cell suspension to the wells of 24-well tissue culture
plates.