Cell Meter™ Flow Cytometric Calcium Assay Kit

Protocol summary

1. Prepare cells in Assay Buffer
2. Add Calbryte™ 520 AM dye-loading solution (1 µL)
3. Incubate at 37°C for 30 minutes
4. Wash the cells
5. Add calcium flux stimulator
6. Monitor fluorescence intensity with flow cytometer using 530/30 nm filter (FITC channel)

Important notes
Thaw all the kit components at room temperature before starting the experiment.

Calbryte™ 520 AM stock solution (500X):
Add 100 µL of DMSO (Not provided) into the vial of Calbryte™ 520 AM stock solution (Component A) and mix them well. Note: 100 µL of Calbryte™ 520 AM stock solution is enough for 100 assays. Unused Calbryte™ 520 AM stock solution can be aliquoted and stored at < -20 °C for more than one month if the tubes are sealed tightly. Protect from light and avoid repeated freeze-thaw cycles.

1. Remove cell culture medium and add 0.5 mL of Assay Buffer. Note: For adherent cells and non-adherent cells, 4 X10⁵ – 8 X10⁵ and 1X10⁶ - 2X10⁶ are recommended to use, respectively. Each cell line should be evaluated on the individual basis to determine the optimal cell density for the intracellular calcium mobilization.
   
   
2. Add 1 µL Calbryte™ 520 AM stock solution (500X) into 0.5 mL non-adherent or adherent cells in Assay Buffer (Component B). Note: If your cells (such as CHO cells) contain organic anion-transports, then probenecid (Component C) may be added to the dye working solution (final in well concentration will be 0.125-1 mM) to reduce leakage of the de-esterified indicators.
   
   
3. Incubate the cells at 37°C for 30 minutes.
   
   
4. For non-adherent cells, centrifuge the cells and remove the dye. Re-suspend the cells in 0.4 mL HHBS (Component D). For adherent cells, use 0.5 mM EDTA to gently lift the cells from the plate and centrifuge. Re-suspend the cells in 0.4 mL HHBS (Component D). Note: For detaching adherent cells from the plate, enzymatic reagents (e.g. trypsin, Accutase) can be considered but need to be tested to make sure the receptor of interest on the cell surface is not affected.
   
   
5. Prepare 5X agonist compound with HHBS or your desired buffer.
   
   
6. Analyze the sample before and after the addition of 100 µL of the prepared agonist on a flow cytometer using 530/30 nm filter (FITC channel). Note: To achieve the best results, it is important to run the assay within 1 minute after the addition of the agonist. It is also important to make sure the time between the agonist addition and the beginning of the actual reading stays constant for all the samples.