Invitrogen™ Fura Red™, AM, cell permeant

The nail polish may be the problem. The K_d value (calcium sensitivity) changes depending upon the dye's environment. Nail polish has solvents that can leech under the coverslip and cause variability. We recommend either going without a sealing or sealing with melted paraffin painted on the coverslip edges with a cotton-tipped applicator (paraffin is hydrophobic and has no solvents).

After loading dye into the cells, intracellular esterases remove the 'AM' moiety from the dye. When the 'AM' group is removed, the dye is able to bind calcium and fluoresce. Since the dye is not covalently bound to any cellular components, it may be actively effluxed from the cell. The rate of efflux is dependent upon the inherent properties of the cell, culture conditions and other factors. The dye may be retained for hours, days or even weeks or lost in a matter of minutes. The use of Probenecid (Cat. No. P36400) limits loss by active efflux.

Unfortunately, we do not have a user manual for the Fura Red, AM, cell permeant (Cat. No. F3021). You can reference section 19.3 of the Molecular Probes handbook for recommendations for use.
There is also a large number of citations & references provided on the product page.

Basically, you can dissolve the product in good quality anhydrous DMSO to prepare a stock solution of 1-10 mM and use a final solution of about 1-10 µM in a physiological buffer. You can then incubate for 15 to 60 minutes, wash and then leave the cells for about 30 minutes to allow the AM group to hydrolyse before starting the assay. You can add Pluronic F-127 (e.g., Cat. No. P6866) to facilitate uptake. Store the DMSO stock solution at -20 degrees C.