Microscopy Media

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 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 - 85ml 220ml 445ml
Stock 10X Stock Powder 10ml 25ml 50ml
Ascorbic Acid Powder 0.0198g 0.0495g 0.099g
NaHCO3 Powder 0.021g 0.0525g 0.105g
BSA Powder 0.15g 0.375g 0.75g
CaCl2 1M Stock 0.15ml 0.375ml 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)

 

top of page BACK TO TOP