Intracellular Calcium[1] 02.09.02

100x Staining Solution

1l Fluo-3 AM (10mg/ml in DMSO) Probes F1241

2l FuraRed AM (10mg/ml in DMSO) Probes F3020

27l Pluronic F-127 (10% in water) Sigma P2443

(i.e. in 1x solution: 3M Fluo-3, 6M FuraRed, 0.1% Pluronic)

 

Cell Stain

1ml Cell suspension

10l 100x Staining solution

45 min in the incubator

1x wash with PBS (4C if possible)

 

Calcium Calibration Curve

Calcium calibration Buffer Kit #1 Probes C-3008

 

Free Ca

Ca free Solution

Ca solution

0M

500.0 L

--

17nM

450.0 L

50.0 L

38nM

444.5 L

55.5 L

65nM

437.5 l

62.5 L

100nM

428.5 l

71.5 L

150nM

416.7 l

83.3 L

225nM

400.0 l

100.0 L

351nM

375.0 l

125.0 L

602nM

333.3 l

166.7 L

 

1L 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

 

 



[1] E.J. Novak and P.S. Rabinovitch, Improved sensitivity in flow cytometric intracellular ionized calcium measurement using fluo-3/Fura Red fluorescence ratios. Cytometry, 1994. 17: 135-41.