Cell Meter™ Cell Viability Assay Kit *NIR Fluorescence Optimized for Fluorescence Microplate Reader*

Protocol summary

1. Prepare cells with test compounds
2. Remove the medium
3. Add CytoCalcein™ NIR working solution (100 µL/well/96-well plate or 25 µL/well/384-well plate)
4. Incubate at room temperature or 37°C for 1 hr
5. Read fluorescence intensity (bottom read mode) at Ex/Em = 635/670 nm (Cutoff = 665 nm) 

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

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

Add 20 µL of CytoCalcein™ NIR stock solution into the bottle of Assay Buffer (10 mL, Component C) and mix well to make CytoCalcein™ NIR working solution. Note: This CytoCalcein™ NIR working solution is for one cell plate and stable for at least 2 hours at room temperature.

Cells Preparation:

Plate 100 to 100,000×10 cells per 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/well/96-well plate, and 25 µL/well/384-well plate. Note: Each cell line should be evaluated on the 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) 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 the medium from the cells. Note: Medium must be removed before loading CytoCalcein™ NIR working solution.
   
   
3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of CytoCalcein™ NIR working solution.
   
   
4. Incubate plate at room temperature or 37°C for 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 = 635/670 nm (Cutoff = 665 nm).