AMPHIBIAN CELL CULTURE, ORGAN CULTURE, AND TISSUE DISSOCIATION

REFERENCE- Antone Jacobson in Methods in Developmental Biology

Each cell has an endogenous food source, so that cultures can be made in simple defined medium. Prepare these solutions.

Holtfreter solution for Ca-free Holtfreter’s, make up the A

Two solutions solution without calcium and add 1 ml

A. B of 0.5 M sodium citrate. For older

3.5g NaCl 20 mg NaHCO3 embryos add 2% trypsin, after boiled.

50 mg KCl 500 ml distilled water bring each solution just to a boil to sterilize it, then

100 mg CaCl2 cool to l7 C in flask with a clean beaker over the top.

500 ml distilled water

(200 mg penicillin 50 mg streptomycin added to A after cooled)

mix the two solutions-Just prior to use, good for one day. (use 1:1 A:B)

Prepare your equipment

1. watchmaker forceps and other tools to be sterilized in cotton lined vessel containing 7O% ethanol- at least ten minutes.

2. flame wax lined dishes'

3. draw out one dozen fine glass needles, if you plan to use parts of embryos., instead of whole ones.

4. boil some water to dip sterile tools In to get rid of alcohol. Keep covered. You can make tools by applying clear nail polish to cue sticks, then attaching a cactus needle.

5. Place embryos In petri dish and sort out good embryos of one stage. Place in Holtfreter’s

solution. Remove jelly vith forceps. Wash 8x with sterile water-embryos.

6. Remove the fertilization membrane so that the embryos are bare, no need to (use

worry about keeping them whole in removing the membranes, it won't hurt it they (Holtfreter

are cut in two. Pipette :into fresh Holtfreter's in a centrifuge tube and store solution

until all are collected (do a dozen). (If parts of embryos are desired, cut out

the desired parts and place than all together In cent, tube.)

7. Use hand centrifuge to pellet the embryos, decant the Holtfreter soln. Add the Ca-free solution with citrate or trypsin added. Gently pull. the embryos up Into a pipette (Boiled to sterilize) and push back out gently to break the cells apart a few times,, then allow to sit in the solution for ten minutes. Pipette the embryos again. If there are no large chunks of embryo left, hand centrifuge the cells down and decant the solution and wash the cells with Ca-free solution no- enzyme),

8. After washing the cells two times, reauspend gently vith a pipette In Holtferter solution and pour into a sterile disposable petri dish. Be sure to cover quickly. Gently agitate periodically and observe reaggregation in your dissecting scope.

9. You can also remove samples with sterile pipettes from time to time., placing them on slides vith vaselined coverslips, observing under high powers recording the number of cells in aggregates.