Intracellular Calcium[1] 02.09.02
100x Staining Solution
1µl Fluo-3 AM (10mg/ml in DMSO) Probes F1241
2µl FuraRed AM (10mg/ml in DMSO) Probes F3020
27µl
Pluronic F-127 (10% in water) Sigma
P2443
(i.e. in 1x solution: 3µM Fluo-3, 6µM FuraRed, 0.1% Pluronic)
Cell Stain
1ml Cell suspension
10µl 100x Staining solution
45 min in the incubator
1x wash with PBS (4°C if possible)
Calcium Calibration Curve
Calcium calibration Buffer Kit #1 Probes C-3008
|
Free Ca |
Ca free Solution |
Ca solution |
|
0µM |
500.0 µL |
-- |
|
17nM |
450.0 µL |
50.0 µL |
|
38nM |
444.5 µL |
55.5 µL |
|
65nM |
437.5 µl |
62.5 µL |
|
100nM |
428.5 µl |
71.5 µL |
|
150nM |
416.7 µl |
83.3 µL |
|
225nM |
400.0 µl |
100.0 µL |
|
351nM |
375.0 µl |
125.0 µL |
|
602nM |
333.3 µl |
166.7 µL |
1µL Calcimycin A23187 (10mM in DMSO) each Fluka 21045
Measurement
|
Flow Cytometer Fluo-3 FL-1 FuraRed FL-3
Fluorescence-Reader Excitation: 485/20nm Emission Fluo-3: 528/20nm Emission FuraRed: 590/35nm (not suitable, EMax at 650nm)
|
from Molecular Probes |
