Multichannel Systems HiClamp Manual - Manual (Page 78)

HiClamp Manual
72
11.3.2 Chemicals
Collagenase
For defolliculation:
 Fresh 1.52 mg/ml collagenase in Barth’s solution without Calcium. The concentration has to be
optimized according to the collagenase batch and experimental conditions, see also chapter
"Defolliculation". Do not prepare solutions in advance as collagenase activity may decrease
rapidly even if the solution is stored at -20 °C.
 Collagenase from Cl. histolyticum ca. 0.17 to 0.28 U/mg (according to Wünsch,1963) lyophilized.
Gentamicin
 Stock solution: 50 μg/ml gentamicin (free base) in Barth’s solution. 1 ml aliquots with 50 mg/ml
gentamicin (free base) are stored at -20 °C.
 Working solution: Dilute 1 ml of gentamicin stock in 1 l Barth's solution.
 Gentamicin sulfate salt, potency approx. 600 μg Gentamicin per mg.
 Alternatively to gentamicin, a mixture of penicillin and streptomycin can also be used to suppress
growth of microorganisms.
Barth’s solution
 pH 7.4 (with NaOH)
 88 mM NaCl
 2.4 mM NaHCO
3
 1 mM KCl
 0.33 mM Ca(NO
3
)2 * 4 H
2
O
 0.41 mM CaCl
2
* 2 H
2
O
 0.82 mM MgSO
4
* 7 H
2
O
 5 mM Tris/HCl
Barth’s solution without Ca 2+
 pH 7.4 (with NaOH)
 88 mM NaCl
 2.4 mM NaHCO
3
 1 mM KCl
 0.82 mM MgSO
4
* 7 H
2
O
 5 mM Tris/HCl
11.3.3 Oocyte Removal
1. Remove the appropriate amount of ovarian tissue surgically from one side of the frog.
Please refer to standard protocols on this subject (Stühmer and Parekh 1995).
2. Transfer the ovarian lobes into a new large Petri or cell culture dish (for example 100 mm
Falcon) filled with Barth’s without Ca 2+ .