Cell Meter™ Cell Viability Assay Kit *Blue Fluorescence*

Protocol summary

1. Prepare cells with test compounds
2. Add the same volume of CytoCalcein™ Blue, AM working solution (100 µL/well/96-well plate or 25 µL/well/384-well plate)
3. Incubate at room temperature or 37°C for 1 hour
4. Monitor fluorescence intensity (bottom read mode) at Ex/Em = 360/450 nm (Cutoff = 420 nm)

Important notes
Thaw one of each kit component at room temperature before starting the experiment.

1. CytoCalcein™ Blue, AM stock solution:
Add 20 µL of DMSO (Component B) into the vial of CytoCalcein™ Blue, AM (Component A), and mix well to make CytoCalcein™ Blue, AM stock solution. Note: 20 µL of CytoCalcein™ Blue, AM stock solution is enough for one plate. Protect from light. For storage, seal tubes tightly.

Add the whole content (20 µL) of CytoCalcein™ Blue, AM stock solution into 10 mL of Assay Buffer (Component C), and mix well to make CytoCalcein™ Blue, AM working solution. This CytoCalcein™ Blue, AM working solution is stable for at least 2 hours at room temperature. Note: If the cells such as CHO cells contain organic-anion transporters which promote the leakage of the fluorescent dye over time, a probenecid stock solution should be prepared and added to the loading buffer at a final in-well working concentration ranging from 1 to 2.5 mM. Aliquot and store the unused probenecid stock solution at ≤ -20 ^oC.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

1. Treat cells with test compounds as desired. Note: It is not necessary to wash cells before adding compounds. However, if tested compounds are serum sensitive, growth medium and serum factors can be aspirated away before adding compounds. Add 100 µL/well (96-well plate) and 25 µL/well (384-well plate) of 1X Hank’s salt solution and 20 mM Hepes buffer (HHBS) or the buffer of your choice after aspiration. Alternatively, cells can be grown in a serum-free media.
   
   
2. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of CytoCalcein™ Blue, AM working solution.
   
   
3. Incubate the plate at room temperature or 37°C for 1 hour, protected from light. (The incubation time could be from 15 minutes to overnight. We got the optimal results with the incubation time less than 4 hours). Note: The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment. Note: DO NOT wash the cells after loading. Note: For non-adherent cells, it is recommended to centrifuge cell plates at 800 rpm for 2 minutes with brake off after incubation.
   
   
4. Monitor the fluorescence intensity with a fluorescence plate reader (bottom read mode) at Ex/Em = 360/450 nm (Cutoff = 420 nm).