Microscopy Buffer (1X)
(pH 7.3; ~320mOsm/kg)
| Component | Final Conc. |
| NaCl | 130.00mM |
| KCl | 5.36mM |
| MgSO4 | 0.80mM |
| Na2HPO4 | 1.00mM |
| Glucose | 25.00mM |
| HEPES | 20.00mM |
| Na-Pyruvate | 1.00mM |
| NaHCO3 | 2.50mM |
| Ascorbic Acid | 1.00mM |
| CaCl2 | 1.50mM |
| BSA | 1.5mg/ml |
Prepare first 10x stock solution:
| 1300.00mM NaCl | 76.00g |
| 53.60mM KCl | 4.00g |
| 8.00mM MgSO4, 7H2O | 1.97g |
| Na2HPO4, 2H2O | 1.97g |
| 250.00mM Glucose | 45.00g |
| 200.00mM HEPES | 47.70g |
| 10.00mM Sodium Pyruvate | 1.10g |
| dWater | to 1L |
 |
Adjust pH to 7.5 with Tris base powder (~9g) |
|
Filter sterilize and store at 4°C |
To make the working buffer:
Component (in order of addition) |
Add from: |
For 100ml |
For 250ml |
For 500ml |
| Water Stock Ascorbic Acid NaHCO3 BSA CaCl2 |
-- 10X Stock Powder Powder Powder 1M Stock |
85ml 10ml 0.0198g 0.021g 0.15g 0.15ml |
220ml 25ml 0.0495g 0.0525g 0.375g 0.375ml |
445ml 50ml 0.099g 0.105g 0.75g 0.75ml |
|
Bring to final volume after all components have been added. |
|
Adjust to pH 7.3 with 1M Tris (If verification of osmolarity is necessary, adjust pH without BSA, then check osmolarity. Add BSA to solution after measurement.) |
|
Filter sterilize |
To prepare a nominally Ca-free buffer:
|
Prepare Working Buffer as above, omitting CaCl2 |
To prepare buffer with 100nM Ca (pCa=7):
|
Prepare Working Buffer as above, omitting CaCl2 |
|
Add 100µM EGTA (20µl of 0.5M EGTA/100ml 1X Buffer) |
