Membrane Potential Assay Kit

Assay Protocol

Kit contents

Each FLIPR Membrane Potential Assay Kit (cat. # R8034) contains the following components and is sufficient for one hundred 96-well or 384-well microplates:

• 1 bottle of 10X Reagent Buffer, Component B (10X Hanks' BSS with 200 mM HEPES, pH 6)
• 10 vials of FLIPR Membrane Potential Assay Reagent, Component A
• Each vial is sufficient for assaying ten 96- or 384-microwell plates

Additional materials needed but not included

• NaOH and HCl, to adjust buffer pH
• 540-590 Bandpass FLIPR Filter Kit from Molecular Devices (cat. #0310-4077)
• Warning: The filter must be installed in the FLIPR system prior to running the assay (see Section 3.1, page 3 of the User Manual).

Storage information

On receipt of the kit, store at room temperature. Under these conditions the reagents are stable for six months in the original packaging. After formulation, the Loading Buffer is stable for up to eight hours and may be stored frozen in aliquots for up to 2 weeks without loss of activity.

Cell handling

The FLIPR Membrane Potential Assay Kit is designed to work in association with many cell types, both adherent and non-adherent. In this section of the protocol we provide guidelines for how to set up the cells for use with the assay kit.

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 Membrane Potential Assay Kit require the creation of a confluent cell monolayer before placing the plates in the FLIPR system.

• 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 poly-D-lysine coated plates. It is recommended that you then centrifuge the plates at 1,000 rpm for 4 minutes.

1. Preparation of loading buffer

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.

   Note: Depending upon the cell type and application, the Hanks/HEPES buffer provided with the FLIPR Membrane Potential Kit may not be the ideal choice. If so, alternative buffers may be used at the discretion of the user in order to achieve optimal results.

2. Remove one vial of Membrane Potential Assay Reagent (Component A).

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

3. 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.

   Prepare the Loading Buffer by diluting the vial mixture in 90 ml of 1X Reagent Buffer. Multiple washes of the vial may be necessary to completely transfer the contents.

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 final volumes are as described. Alternatively, cells can be grown in serum-free conditions.

   Warning: do NOT wash the cells after dye loading.

2. Incubate cell plates at 37 °C for 30 minutes.

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

3. Running the FLIPR membrane potential assay

1. Before incubation, remove the filter holder located inside the FLIPR system's filter door. Briefly, release the two thumbscrews holding the filter holder in place and slide the holder out onto a clean or towel-lined bench top. Filter #2 location should be empty. Remove one ring by unscrewing in a counter-clockwise direction. Carefully place the 540-590 bandpass emission filter in the #2 location and screw the ring back in place with the notches facing outward. Place the filter holder in its correct position in the FLIPR system.
2. Choose Filter #2 in the experimental setup of the FLIPR software. After incubation, transfer the plates directly to the FLIPR system and begin the Membrane Potential Assay. The Membrane Potential Assay may be run at room temperature up to physiological temperature.
3. Recommended experimental setup parameters are as follows. Note that the addition rates 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 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