Materials 21 Solutions

A variety of saline solutions are commonly used to culture amphibian embryonic tissues (63). Our favorites are modified Barth's solution (MBS) (64), a general-purpose saline, and modified Danilchik's solution (DFA), a saline specialized for supporting the normal motility and behavior of deep, nonepithelial cells (65).

MBS (modified Barth's solution, from Gurdon, ref. 64):

Stock Per 1 L Final concentration, mM

50 mM MgSO4 16.4 mL 0.82

0.8 M NaHCO3

3.0 mL

2.40

0.1 M KCl

10.0 mL

0.01

33 mM Ca(NO3)2

10.0 mL

0.03

1 M CaCl2

0.41 mL

0.41

Hepes

2.38 g/L

5.00

Adjust pH to 7.4 with NaOH.

DFA ("Danilchik's for Amy" from Sater et al., ref. 65):

Stock

In 500 mL

In 1 L

Final concentration

4 M NaCl

6.625 mL

13.25 mL

53.0 mM

1 M Na2CO3

2.5 mL

5.0 mL

5.0 mM

K gluconate

0.525 g

1.05 g

4.5 mM

Na gluconate

3.49 g

6.93 g

32.0 mM

1 M CaCl2

0.5 mL

1.0 mL

1.0 mM

1 M MgSO4

0.5 mL

1.0 mL

1.0 mM

Adjust pH to 8.3 with 1 M bicine.

Adjust pH to 8.3 with 1 M bicine.

The original Danilchik's (66,67) as well as subsequent versions (see refs. 39,65) were developed to mimic the ionic composition of the blastocoel fluid of X. laevis (68).

Embryos are kept in third-strength saline, and then transferred to full-strength for microsurgery. DFA is the appropriate solution for "open-faced" explants, which contain exposed deep cells. Although DFA allows more normal behavior of deep cells, it interferes with some functions of the epithelial cells, such as neural fold fusion (see refs. 11,66,67). MBS or an equivalent should be used for all other microsurgery. After healing, the embryos should be transferred to 3rd- or 10th-strength saline. We make two types of full-strength salines, one plain and one with 0.1% bovine serum albumin (BSA). BSA reduces adhesion of the cells to the surfaces of the dish and coverslips. Embryos should be transferred to plain saline before fixation, or the BSA may be fixed or precipitated on the surface of the specimens, making them appear dirty in a number of staining procedures. Solutions are filtered through a 0.22-|m Millipore filter, and aliquoted into 50-mL plastic centrifuge tubes, and frozen at -20oC until needed. We also freeze 1.0-mL aliquots of 100X antibiotic/antimycotic solution (10,000 U penicillin, 10 mg streptomycin, 25 |g amphotericin B, per mL, in 0.9% NaCl, Sigma Chemical, St. Louis, catalog #A9909) at -20oC. These are thawed and added at 0.5 mL/50 mL tube of solution at the time of use. Culture solutions are replenished every 12 h. Embryos are kept at

16-24oC. We try to avoid temperatures below or above this range, though two more degrees in either direction may be fine with good spawnings.

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