Calcium Flux Measurement Using Indo-1

1. Dissolve the contents of 1 vial (50 µg indo-1) in 50 µl dry DMSO.  This makes a 1mM solution.  T and B cells can be loaded with 1 mM indo-1; granulocytes or monocytes with 1-3 mM and dendritic cells with 3-6 mM Indo-1.  Cell concentration on loading should be 1-10 x 106/ml. 
   
   
2. Incubate cells at 37°C for 45-60 min in a waterbath or incubator.  Wash twice in serum free medium or PBS.  Resuspend at 10e6/ml in PBS or serum free medium of choice.  Either keep the cells at 37°C if using immediately or keep on ice until ready for use; prior warming back to RT or 37°C should be done as Indo-1 fluorescence is temperature dependent. 
   
   
3. Immunophenotyping if required should then be carried out on ice.
   
   
4.  Cells can then be analysed on BD LSR which allows up to six-colour analysis.  Thus FITC, PE, TC and APC can be used for immunophenotyping.  Indo-1 is only loaded into viable cells thus there is no requirement to exclude dead cells by PI.
   
   
5. The filter set-up on the BD LSR for Indo-1 (UV excitation only) is for calcium bound Indo-1 violet FL-5 424/44 nm BF filter and unbound Indo-1 green FL-4 530/30nm BF filter.  Calcium flux is measured as a Ratio between calcium bound Indo-1 and unbound or FL-5/FL-4 versus time. 
   
   
6. Full scale deflection of the calcium flux is measured by the addition of ionomycin 1-10 mg/ml.  Contribution of internal stores of calcium can be measured by resuspending cells in calcium-free medium and then adding ionomycin.