FLIPR Calcium 3 Assay Kit

Troubleshooting Guide

1. Fluorescence drop upon compound addition

This may result from dislodging cells from the wells during addition. Lowering the addition speed (RATE) to 10-60 µl/sec for FLIPR® or to '1' for FlexStation™ should solve the problem.

Adding volumes greater than recommended may increase the initial fluorescence drop. In these cases it may be necessary to adjust the volumes of the components. The recommended volume of the Loading buffer is 100 µl for 96-well plates and 25 µl for 384-well plates.

Warning: Increasing the final in-well concentration of the Loading Buffer may decrease the response of the assay. If only one addition is required, then add the appropriate volume of buffer for Addition 1.

2. Serum-sensitive cells or targets

Some cells are serum-sensitive resulting in oscillations of intracellular calcium that could interfere with results. Also, some target receptors or test compounds may interact with serum factors. In these cases, the growth medium should be removed (and the cells may also need to be washed) prior to addition of loading buffer. The volume of growth medium removed should be replaced with an equal volume of 1X HBSS buffer before loading. Alternatively, the cells could be incubated overnight in lower concentrations of FBS and not washed prior to the addition of Dye Loading Buffer.

3. Cells tested with buffer plus DMSO show a calcium response

The buffer used for the negative control wells should contain the same final concentration of DMSO as is present in the wells containing the test compounds. However, this concentration of DMSO could cause a calcium flux. In these cases, add DMSO to the Loading Buffer such that the final concentration of DMSO in the wells does not change after buffer addition.

4. Precipitation in the reagent buffer

The Calcium 3 Assay Kit is compatible with numerous buffers. Use buffers shown to work in previously established assays, if available.

5. Response is smaller than expected

The agonist and antagonist may stick to the tips and trays. Use 1% BSA in all compound buffer diluents and presoak tips compound buffer/1% BSA.

Warning: Do not use the same compound plate for presoaking and compound addition when using a 384 Pipettor head in the FLIPR System. Instead, use a 'boat' for presoaking.

6. Apparent well-to-well variation is observed

A Multidrop® or equivalent dispenser is recommended for use with all additions off the FLIPR or FlexStation if apparent well-to-well variation is observed. Add a single mix cycle using 15 µl to the FLIPR or FlexStation addition protocol.

In some cases allowing the plates to stand at room temperature prior to use in the assay may decrease well-to-well variation.