Cell Meter™ Cell Viability Assay Kit *Green Fluorescence*

Protocol summary

1. Prepare cells with test compounds
2. Add the same volume of working solution as the cell medium
3. Incubate at room temperature or 37°C for 1 hour 
4. Monitor fluorescence with fluorescence microplate reader at Ex/Em= 490/525 nm (Cutoff=515 nm)

Important notes
Thaw one of each kit component at room temperature before use.

1. CytoCalcein™ Green stock solution:
Add 20 µL of DMSO (Component B) into the vial of CytoCalcein™ Green (Component A) and mix well. Protect from light. Note: 20 µL of CytoCalcein™ Green stock solution is enough for one plate.

Add the whole content (20 µL) of CytoCalcein™ Green stock solution into 10 mL of Assay Buffer (Component C) and mix well. Note: The 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 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.

Cells Preparation:

Plate 100 to 100, 000 cells/well in a tissue culture microplate with black wall and clear bottom. Add test compounds into the cells and incubate for a desired period of time (such as 24, 48 or 96 hours) in a 37 °C, 5% CO₂ incubator.

For blank wells (medium without the cells), add the same amount of compound buffer. The suggested total volume is 100 µL for a 96-well plate, and 25 µL for a 384-well plate. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for proliferation or cytotoxicity induction. For proliferation assays, use fewer cells; for cytotoxicity assays, use more cells to start with.

Sample Protocol:

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) or 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. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of dye-loading solution.
   
   
3. Incubate the dye-loading 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 at Ex/Em = 490/525 nm with cutof=515 nm.