CHAPTER 6 - DISSOCIATED CELL CULTURE
Contents
Preparing Embryos for Cell Culture
0 Ca2+ - Mechanical Dissociation
Protease Dissociation
Dissociation of Embryos by Dissection
Using Coated Glass Coverslips for Cell Culture
Recipes:
Embryo Extract
Growth Medium
CHAPTER 6 - DISSOCIATED CELL CULTURE
(Source: D. Frost and D. Sepich)
Treat embryos with bleach (0.5% solution for 2 min) before removing the chorions. Decant the bleach solution and rinse with 10% sterile Hank's saline. Remove the chorions with watchmaker's forceps or by soaking the embryos in pronase (see Removing Embryos from Chorions, Chapter 4). Embryos that can move should be anesthetized by soaking in tricaine or by chilling on ice. Embryos can be dissociated with 0 Ca2+ up to about 16 h of development. Older animals should be dissociated with the protease treatment or by dissection.
1. Rinse embryos in sterile, calcium-free Ringer's solution.
2. Transfer about 10-100 embryos in a single drop of calcium-free Ringer's solution to the center of a tissue culture plate.
3. Drop a sterile coverslip onto the embryos and smash them.
4. Add more calcium-free Ringer's solution, remove the coverslip and suspend the cells by swirling the plate.
5. Transfer the suspension to a sterile centrifuge tube and spin at 300 x g for 7 min.
6. Remove most of the supernatant, triturate the pellet through a sterile narrow-bore Pasteur pipette to resuspend the cells and then add several ml of growth medium.
7. Centrifuge, 300 x g, 7 min.
8. Remove supernatant and resuspend in enough growth medium to make a final concentration of 15 fish per ml.
9. Plate the cells and incubate at 28.5°C.
1. Rinse embryos in sterile, calcium-free Ringer's solution, 15 min.
2. Transfer the embryos to a dish containing Custom ATV solution (Irvine Scientific) or 0.25% trypsin, 1 mM EDTA, pH 8.0 in sterile PBS. Incubate at 28.5°C and monitor the dissociation with a microscope. Intermittently triturate with a sterile, narrow-bore pasteur pipette and continue until you see mostly single cells.
3. Add CaCl2 to 1-2 mM and fetal calf serum to 5-10% to stop the reaction.
4. Centrifuge at 100-300 x g for 3 min.
5. Discard the supernatant and resuspend the cells in L-15 (Sigma) supplemented with 0.3 mg/ml glutamine, 50 U/ml penicillin, 0.05 mg/ml streptomycin, and 0.8 mM CaCl2.
6. Repeat step 4 and resuspend in supplemented L-15 (as in step 5) with 10% embryo extract and 3% fetal calf serum to make a final concentration of 15 embryos per ml.
7. Plate cells on plastic or coated glass and incubate at 28.5°C without additional atmospheric CO2.
1. Place embryos into high calcium Ringer. Cut or pull into pieces using sharpened forceps or needles.
2. Rinse by dipping into growth medium twice.
3. Plate onto tissue culture dish in a single drop of medium with 5% serum (to help attachment).
4. Incubate 4 hr at 28.5°C.
5. Gently fill the dish with growth medium without serum.
Zebrafish embryonic cells attach well to glass coated with laminin or to plastic. They don't attach well to bare glass or glass coated with fibronectin. They attach somewhat to 0.2 mg/ml gelatin coated glass.
1. Sterilize glass coverslips (22 mm x 22 mm) by dipping in ethanol and flaming.
2. Place a drop of 10 µg/ml laminin (Boehringer Mannheim) in sterile PBS and leave for 45 min.
3. Rinse thoroughly with sterile PBS or dH2O.
4. Place a drop of cell suspension (cells from 1-4 embryos) onto a 0.5-1.0 cm spot in the center of the coated glass coverslip in a petri dish and allow the cells to attach for several hours or overnight.
5. Flood the dishes with growth medium.
1. Chill 200 3 d embryos after removing from chorions.
2. Rinse in 0.5% chilled bleach for 2 min and then in zero calcium Ringer for 2 min.
3. Transfer to a Dounce homogenizer with a minimum of liquid and homogenize well.
4. Resuspend in 1 ml L-15 supplemented with 0.3 mg/ml glutamine, 50 U/ml penicillin, 0.05 mg/ml streptomycin and 0.8 mM CaCl2.
5. Store at -20°C.
L-15 (Sigma), 0.3 mg/ml glutamine, 50 U/ml penicillin, 0.05 mg/ml streptomycin, 0.8 mM CaCl2, 10% embryo extract, and 3% fetal calf serum. Filter through a syringe filter.
