FLIPR Calcium Assay Kit

Troubleshooting Guide

1. Fluorescence drop upon compound addition This may be the result of dislodging cells from the wells during addition. Lowering the addition speed to 10-60 µl/sec should solve the problem in this case. If a lower addition speed eliminates the drop in fluorescence, then seed fewer cells in the wells to avoid this problem in future. If a fluorescence drop is still present after lowering addition speed, then it may be the result of a dilution effect. Using the Loading Buffer at a higher concentration (as in the parameters in the table below) has been shown to work in this case.

Parameters	96-well Plate	384-well Plate
Dilution volume for loading buffer	50 ml	66 ml
Addition volume for loading buffer	50 µl	25 µl
Compound concentration (fold)	4X	4X
Addition volume for compound (total for agonist and antagonist)	50 µl	17 µl

2. Different compound concentration

The concentration of the Loading Buffer may be adjusted to compensate for compounds that are at different concentrations from that recommended in the table in Section 3.2 of the Calcium Kit Assay Protocol. The following table is provided as a guideline.

Note: It is not recommended to dilute the Loading Buffer to less than 33 ml. The Loading Buffer may become too viscous for proper handling.

	Compound		Loading Buffer
Plate	Addition Volume (Agonist and Antagonist)	Concentration (Fold)	Dilution Volume	Addition Volume
96-well	67 µl	3X	33 ml	33 µl
	50 µl	4X	50 ml	50 µl
384-well	17 µl	4X	66 ml	25 µl
	25 µl	3X	66 ml	25 µl

3. 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 Reagent Buffer before loading. Alternatively, the cells could be incubated overnight in lower concentrations of FBS (i.e., 0.5%) and not washed prior to the addition of Loading Buffer.

4. 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 so that the final concentration of DMSO in the wells will not change after buffer addition.

5. Precipitation with the Loading Buffer

The FLIPR Calcium Assay Kit is compatible with numerous buffers. If the Loading Buffer prepared with 1X Reagent Buffer causes precipitation (or some other unexpected effect), prepare the Loading Buffer with a different buffer that has been shown to work in previously established assays.

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