Cell Meter™ Cell Viability Assay Kit *Blue Fluorescence with 405 nm Excitation*
|Shipping||Standard overnight for United States, inquire for international|
|H-phrase||H303, H313, H333|
|Intended use||Research Use Only (RUO)|
|R-phrase||R20, R21, R22|
Fluorescence microplate reader
|Recommended plate||Black wall/clear bottom|
|Instrument specification(s)||Bottom read mode|
AT A GLANCE
- Prepare cells with test compounds
- Add the same volume of CytoCalcein™ Violet 450, AM working solution (100 µL/well/96-well plate or 25 µL/well/384-well plate)
- Incubate at room temperature or 37°C for 1 hour
- Monitor fluorescence intensity (bottom read mode) at Ex/Em = 405/460 nm (Cutoff = 435 nm)
Thaw one of each kit component at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. CytoCalcein™ Violet 450, AM stock solution:
Add 20 µL of DMSO (Component B) into the vial of CytoCalcein™ Violet 450, AM (Component A), and mix well to make CytoCalcein™ Violet 450, AM stock solution. Note: 20 µL of CytoCalcein™ Violet 450, AM stock solution is enough for one plate. Protect from light. For storage, seal tubes tightly.
PREPARATION OF WORKING SOLUTION
Add the whole content (20 µL) of CytoCalcein™ Violet 450, AM stock solution into 10 mL of Assay Buffer (Component C), and mix well to make CytoCalcein™ Violet 450, AM working solution. This CytoCalcein™ Violet 450, 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 °C.
For guidelines on cell sample preparation, please visit
SAMPLE EXPERIMENTAL PROTOCOL
- 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.
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of CytoCalcein™ Violet 450, AM working solution.
- 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.
- Monitor the fluorescence intensity with a fluorescence plate reader (bottom read mode) at Ex/Em = 405/460 nm (Cutoff = 435 nm).
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