- Solutions for Cell-Attached and Excised
Inside-Out
- Recording Configurations
A. RECORDING PIPETTE SOLUTIONS
1. Standard Saline (150 mM NaCl), 500 mL
| |
MW |
mM |
g |
| NaCl |
58.44 |
150 |
4.38 |
| KCl |
74.55 |
3 |
0.11 |
| HEPES |
238.31 |
10 |
1.19 |
| glucose |
180.16 |
14 |
1.26 |
| CaCl2 |
110.99 |
2 |
0.11 |
| MgCl2 |
95.23 |
1 |
0.05 |
Titrate with NaOH to pH=7.4 before you add divalents. This prevents formation
of insoluble precipitates.
- TEA
- TEA blocks Ca2+-activated K+ channels (75% block at 1 mM). TEA produces fast block of neuronal and skeletal muscle nicotinic receptors at µM concentrations (Marshall et al, 1990; Nojima et al., 1993). TEA also blocks NMDA receptors. TEA does not block Ca channels and is used to block outward K+ currents that can obscure Ca channel activity.
- K(ATP) Channels
- Saline for recording K(ATP) channels should be made up without Mg,
which blocks the channel (Davies, 1990). Use 75 mM NaCl and 75 mM KCl in
order to look at inward K currents (see Davies et al, 1989; Davies, 1990).
- 110 mM Ba (Hess et al., 1986; Lansman, 1990)
- pH=7.4, 271 mOsm
- In mM: 110 BaCl2, 10 glucose, 10 HEPES. Titrate with TEA-OH to pH=7.4. Add barium last after titration to prevent precipitation of insoluble hydroxides.
- 150 mM Li (Hess et al., 1986)
- In mM: 150 LiCl, 35 glucose, 10 HEPES, 5 EDTA. Titrate with TEA-OH
to pH=7.4
- 90 mM Ba Solution
- For recording Ca channels in cerebellar granule cells (Slesinger and Lansman, 1991b). In mM: 90 BaCl2, 15 TEA-Cl, 10 HEPES, 10 glucose. Titrate with TEA-OH to pH 7.4. Add BaCl2 last to prevent precipitation of insoluble hydroxides.
- 150 mM CsCl Solution
- For NMDA Channels in Outside-Out Patches, 500 mL (adapted from Jahr
and Stevens, 1990)
| |
MW |
mM |
g |
| CsCl |
168.37 |
150 |
12.63 |
| HEPES |
238.31 |
5 |
0.60 |
| EGTA |
380.4 |
10 |
1.90 |
| MgATP |
531.5 |
2 |
0.53 |
Titrate with CsOH to pH=7.3. 336 mOsm
- 140 mM K-Methanesulfonate
- For neuronal K Channels, 500 mL. May be used both in the pipette and
as a depolarizing bath solution for cell-attached and inside-out patches.
| |
MW |
mM |
g |
| NaCl |
58.44 |
10 |
0.29 |
| KOH |
56.11 |
140 |
3.93 |
| HEPES |
238.31 |
10 |
1.19 |
| EGTA |
380.4 |
5 |
0.95 |
| glucose |
180.16 |
90 |
8.11 |
| MgCl2 |
95.23 |
2.5 |
0.12 |
| TEA (optional) |
165.71 |
10 |
0.83 |
Titrate with methanesulfonic acid to pH=7.4. The glucose concentration
was empirically determined to raise osmolarity to ~315 mOsm. Reduce glucose
to adjust osmolarity if adding TEA-Cl.
B. BATH SOLUTIONS
- +(S)-202-791 Dihydropyridine Agonist Stock Solution
- +(S)-202-791 is the enantiomer of Bay K8644 which opens L-type Ca channels
for long periods. Make up a 10 mM stock solution in 100% ethanol and store
in the freezer in a light-proof container. Dilute into 4 ml bath solution
for use in experiments. Add 4 µl of the 10 mM stock solution to 4
ml bath solution to make a 10 µM working solution. The working solution
should be made fresh daily. Add 100 µl of 10 µM working solution
to the culture dish containing ~900 µl bath solution to achieve a
final concentration of ~1 µM. To achieve complete diffusion of the
agonist, the stock volume should be at least 10% of the volume of the bath.
Dihydropyridines are best used with disposable plasticware. Glassware requires
extensive cleaning and exposure to UV light to remove all traces of the
drug.
150 mM K-aspartate Depolarizing Solution, 500 mL (Hess et al., 1986;
Lansman, 1989)
| |
MW |
mM |
g |
| aspartic acid |
133.10 |
150 |
9.98 |
| KOH |
56.10 |
150 |
4.21 |
| HEPES |
238.31 |
10 |
1.19 |
| glucose (to adjust osmolarity) |
180.16 |
4 |
0.36 |
| EGTA |
380.4 |
1 |
0.19 |
| MgCl2 |
95.23 |
5 |
0.24 |
Ca is omitted to prevent excess Ca influx under depolarization. MgCl2 promotes membrane stabilization.
- 150 mM K-Methanesulfonate Depolarizing Solution (Slesinger and Lansman,1991b)
- In mM: 150 KOH, 1 EGTA, 10 HEPES, 60 glucose, 5 MgCl2. Titrate with methanesulfonic acid to pH 7.4.
- 125 mM K-Mes Depolarizing Solution for Cerebellar Granule Cells
(Inside-Out)
- In mM: 125 KOH, 10 NaCl, 5 EGTA, 10 HEPES, 20 glucose, 2.5 MgCl2. Titrate with methanesulfonic acid to pH 7.4
- 150 mM Na Solution for NMDA Channels in Outside-Out Patches, 500
mL
- (adapted from Jahr and Stevens, 1990)
| |
MW |
mM |
g |
| NaCl |
58.44 |
150 |
4.82 |
| HEPES |
238.31 |
5 |
0.60 |
| glucose |
180.16 |
20 |
1.80 |
| CaCl2 |
110.99 |
2 |
0.11 |
Titrate with NaOH to pH=7.3. 336 mOsm.
References