KineticBlue™ Fluorometric Cell Viability Assay

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Catalog Number : CC1100
Description
KineticBlue™ Fluorometric Cell Viability Assay Kit offers a simple, rapid, reliable, sensitive, safe and cost-effective measurement of cell viability. Kinetic Blue™ detects cell viability by utilizing a blue and nonfluorescent dye resazurin, which is converted to a pink and fluorescent dye resorufin in response to chemical reduction of growth medium resulting from cell growth. Continued cell growth maintains a reduced environment while inhibition of growth maintains an oxidized environment. Reduction related to growth causes the REDOX indicator to change from the oxidized (nonfluorescent, blue) form to the reduced (fluorescent, red) form. The fluorescent signal is monitored using 530-560 nm excitation wavelength and 590 nm emission wavelength. The absorbance is monitored at 570 nm and 600 nm. For optimal result, subtract background OD at 600 nm from OD at 570 nm. The fluorescent and colorimetric signal generated from the assay is proportional to the number of living cells in the sample. The KineticBlue™ assay is designed to quantitatively measure the proliferation of various human and animal cell lines, bacteria and fungi.

KineticBlue™ Cell Viability Assay.

This assay  is as sensitive as thymidine assay for detecting cell proliferation. Depending on the cell types, Kinetic Blue™ can detect as few as 40 cells with reproducible and sensitive signal. Considering resorufin (pink and fluorescent) can be further reduced to hydroresorufin (uncolored and nonfluorescent), the assay signal decreases even with increased number of cells after all resazurin is converted into resorufin. Therefore, it is important to conduct a cell number titration assay for each particular cell line of your interest to identify the optimal number of cells for your assay to avoid this potential pitfall.

Storage and Handling
Upon receipt, the kit should be stored at 4°C and protected from light. Stored properly, the kit components should remain stable for 6 months.

• Plate cells in 100µL medium into 96-well tissue culture plates by conducting cell number titration in the range of 40 to 10,000 for adherent cells and 2,000 to 500,000 for suspension cells. For background control, use 100µL medium without cells.
• Add 10µL KineticBlue^TM solution into medium and incubate cells at 37^oC overnight.
• Measure absorbance at 570 nm and 600 nm or fluorescence with excitation wavelength at 530 nm and emission wavelength at 590 nm using a micro-titer plate reader.
• Obtain OD570-OD600 for each sample if colorimetric detection method is chosen, or fluorescence signal from each sample deducted by background fluorescence from the background control, and plot a standard curve to identify the optimal cell concentration for your assay.
• Plate cells into 96-well tissue culture plates using optimal cell concentration.
• Carry out your experiment by adding agents of your interest into appropriate well and incubate with cells for a certain period of time.
• Add 10µL KineticBlue^TM solution into medium and incubate cells at 37^oC overnight.
• Measure absorbance at 570 nm and 600 nm or fluorescence with excitation wavelength at 530 nm and emission wavelength at 590 nm using a micro-titer plate reader.
• Obtain OD570-OD600 for each sample if colorimetric detection method is chosen, or fluorescence signal from each sample deducted by background fluorescence from the background control.

Ahmed SA, Gogal RM Jr, Walsh JE. A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H] thymidine incorporation assay. J Immunol Methods. 1994 Apr 15;170(2):211-24.

Shahan TA, Siegel PD, Sorenson WG, Kuschner WG, Lewis DM. A sensitive new bioassay for tumor necrosis factor. J Immunol Methods. 1994 Oct 14;175(2):181-7.

Nociari MM, Shalev A, Benias P, Russo C. A novel one-step, highly sensitive fluorometric assay to evaluate cell-mediated cytotoxicity. J Immunol Methods. 1998 Apr 15;213(2):157-67.

Kinetic Blue™ is a registered trademark of Krishgen Biosystems.