FLIPR Calcium Assay Kit

Assay Protocol

cell handling Protocol PDF  [PDF] For adherent cells, we recommend seeding cells overnight with a plating volume of 100 µL/well for 96-well plates or 25 µL/well for 384-well plates. For non-adherent cells, we recommend centrifugation of the cells from culture medium and suspension of the pellet in culture medium. Add 100 µL (96-well plate) or 25 µL (384-well plate) of cell suspension to each well of the plate. It is recommended that you then centrifuge the plates at 1000 rpm for up to 4 minutes.

1. Preparation of loading buffer

The FLIPR Calcium Assay Kit is designed to work in association with many cell types, both adherent and non-adherent. In this section we provide guidelines for how to set up the cells for use with the assay kit. For a current list of cells and receptors successfully used with the FLIPR Calcium Assay Kit, contact your MDC technical support representative or click here.

We recognize that a variety of cell handling conditions might be adopted at the discretion of the user based on standard operating procedures in the laboratory. Optimal cell conditions for the FLIPR Calcium Assay Kit require the creation of a confluent cell monolayer before placing the plates in FLIPR. In general, we recommend starting with 50,000 cells/well for a 96-well plate and 10,000 cells/well for a 384-well plate.

The following procedure is designed for 10 (ten) 96- or 384-well plates using either adherent or non-adherent cells prepared as described above.

1. To prepare the 1X Reagent Buffer, pipette 10 mL of 10X Reagent Buffer (Component B) and dilute to 100 mL with distilled water. Adjust to pH 7.4 with NaOH.

   Note: occasionally a white precipitate will form in the 10X Reagent Buffer bottle. This is normal and will not affect the assay.

2. Remove one vial of FLIPR Calcium Assay Reagent (Component A) and equilibrate to room temperature.

   Dissolve contents of vial completely by adding 10 mL of 1X Reagent Buffer. Mix by repeated pipetting until the contents are completely dissolved.

3. Prepare the Loading Buffer by diluting the vial mixture in 90 mL of 1X Reagent Buffer. Multiple washes of the vial are necessary to completely transfer the contents.

   Note: if your cells require probenecid, then a stock solution should be prepared fresh and added fresh to the Loading Buffer at a final in-well working concentration of 2.5 mM. Do not store frozen aliquots of Loading Buffer with probenecid and always add fresh probenecid the day of the experiment.

   Warning: the components supplied are sufficient for proper cell loading. For optimum results it is important NOT to add any additional reagents or change volumes and concentrations, except as indicated in the Troubleshooting Guide.

2. Loading cells using loading buffer

1. Remove cell plates from the incubator or centrifuge. Do not remove the supernatant. Add an equal volume of Loading Buffer to each well (100 µL per well for 96-well plates, 25 µL for 384-well plates).

   Although Molecular Devices does not recommend washing cells before dye loading, growth medium and serum factors can be washed away before adding the Loading Buffer, provided residual volumes after the wash step are as described. Alternatively, cells can be grown in serum-free conditions.

2. Incubate cell plates for 1 hour at 37 °C.

   Note: in some cases, incubation at room temperature may work best.

   Note: the FLIPR Calcium Assay Kit is optimized for addition of antagonists as well. For 96-well plates add 50 µL to each well and for 384-well plates add 25 µL. Addition volumes of agonist described below do not change, but fold concentrations and pipettor height do change.

   Warning: do NOT wash the cells.

3. Running the FLIPR calcium assay

1. After incubation transfer the plates directly to FLIPR and begin the calcium assay as described in the FLIPR system manual.
2. Recommended experimental setup parameters are as follows. Note that the addition speeds are faster than in the conventional protocol because of the increased robustness of the cells after the new loading procedure.

   Faster addition speeds can lead to better mixing of compounds and lower signal variance across the plate.

Parameters	96-well Plate	384-well Plate
Addition Volume (µL)	50	25
Compound Concentration (Fold)	5X	3X
Addition Speed (µL/sec) Adherent Cells	50-100	10-20
Addition Speed (µL/sec) Non-adherent Cells	10-20	5-10