实验概要
Measure the calcium in cytosol with indo-1.
主要试剂
DMSO, Imonomycin, RPMI1640, PBS
实验步骤
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 μM indo-1; granulocytes or
monocytes with 1-3 μM and dendritic cells with 3-6 μM Indo-1. Cell
concentration on loading should be 1-10 x 106/ml.
2. Incubate cells at 37°Cfor 45-60 min in a waterbath or incubator. Wash twice in serum free medium or PBS. Resuspend at 106/ml
in PBS or serum free medium of choice. Either keep the cells at 37°Cif
using immediately or keep on ice until ready for use; prior warming back
to RT or 37°Cshould 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 μg/ml.
Contribution of internal stores of calcium can be measured by
resuspending cells in calcium-free medium and then adding ionomycin.