Cell Meter™ Cell Viability Assay Kit *Red Fluorescence*

Protocol summary

1. Prepare cells with test compounds
2. Remove the medium
3. Add CytoCalcein™ Red AM working solution (100 µL/well/96-well plate or 25 µL/well/384-well plate)
4. Incubate at room temperature or 37°C for 30 minutes - 1 hr
5. Monitor fluorescence intensity (bottom read mode) at Ex/Em = 540/590 nm (Cutoff = 570 nm) 

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

1. CytoCalcein™ Red AM stock solution:
Add 20 µL of DMSO (Component B) into the vial of CytoCalcein™ Red AM (Component A) and mix well to make CytoCalcein™ Red AM stock solution. Note: 20 µL of CytoCalcein™ Red AM stock solution is enough for one plate. Protect from light. For storage, seal tubes tightly. Note: Unused CytoCalcein™ Red stock solution can be aliquoted and stored at < -20 ^oC for one month if the tubes are sealed tightly. Avoid repeated freeze-thaw cycles.

Add the whole content (20 µL) of CytoCalcein™ Red AM stock solution into 10 mL of Assay Buffer (Component C), and mix well to make CytoCalcein™ Red AM working solution. Note: This CytoCalcein™ Red AM working solution is not stable, use it promptly. Note: If the cells, such as CHO cells, contain organic-anion transporters which cause 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 of 1-2.5 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 compound. 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 serum-free media.
   
   
2. Remove growth medium.
   
   
3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of CytoCalcein™ Red AM working solution.
   
   
4. Incubate plate at room temperature or 37°C for 30 minutes to 1 hour, protected from light. Note: The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment. For non-adherent cells, it is recommended to centrifuge cell plates at 800 rpm for 2 minutes with brake off after incubation.
   
   
5. Monitor the fluorescence intensity with a fluorescence plate reader (bottom read mode) at Ex/Em = 540/590 nm (Cutoff = 570 nm).