Traditional cell counting methods take time and effort to carry out for any meaningful number of samples, making them unfeasible for large scale / high-throughput experiments such as screening methods. Spectrophotometric cell counting methods, however, due to their use of absorbance / fluorescence / chemiluminescence measurement as a direct representation of relative cell number, allow for high-precision results to be obtained with minimal time and effort on the part of the experimenter. Here at TCI, we offer all three: an absorbance-based cell counting reagent (Product No. M3353), a fluorescence-based cell counting reagent (Product No. R0195), and a chemiluminescence-based cell counting reagent (Product No. A3495 and A3519) to suit your individual needs.
ATP-Luciferase Cell Viability Assay Solution (for Chemiluminescence Detection)
ATP-Luciferase cell viability assay solution (Product No. A3519 and A3495) allows for the quick (on the order of 10 minutes) measurement of large numbers of samples at once.
This reagent is based on the chemistry of the firefly luciferase reaction. Luciferase oxidizes its substrate luciferin in the presence of ATP and molecular oxygen, giving off light of roughly 560nm in the process. The linearity of this luminescence is preserved over a wide range of ATP concentrations, and can be used as an indicator of relative ATP concentration over this range. As the intracellular concentration of ATP does not vary significantly over the course of the cell cycle, the luciferase reaction, used to determine intracellular ATP concentration, can be used to indirectly measure relative cell numbers.1,2) In the case of this reagent linearity is preserved for between 20 and 10000 cells per well of a 96-well plate.
Measurement of chemiluminescence means no special filters are required. Additionally, this reagent, when added directly to culture medium, produces a color change from red to yellow that can be used as a visual guide to prevent double-dispensing.
- Thaw appropriate volume of ATP-luciferase cell viability assay solution (Product No. A3495 or A3519) on ice.
- Add volume of thawed assay reagent equivalent to volume of culture medium in each well.
- Pipette gently to mix, and incubate at RT for 10 minutes.
- Measure chemiluminescence on an appropriate instrument.
MTT Solution (for UV Absorbance Detection)
MTT is a type of tetrazolium salt that is converted into formazan when taken up by living cells. This formazan can be dissolved in DMSO and the absorbance at 540 nm can be used to estimate the relative number of cells. MTT solution (Product No. M3353) consists of 5.0 mg/mL MTT dissolved in PBS.
Application: Cell proliferation assay
- Seed 100 μL of cells in a 96-well plate, and Incubate overnight in an incubator.
- Add 10 μL of MTT solution (Product No. M3353) to each well.
- Incubate for 2-4 hours until a color change is seen.
- Remove medium, taking care not to inhale the formazan.
- Add 100 μL of DMSO and dissolve the formazan.
- Measure the absorbance at 540 nm.
*Please adjust staining time and volume as needed. Preliminary tests should be performed.
Resazurin Solution (for Fluorescence Detection)
Resazurin can be used to determine relative cell number, useful in the quantification of such phenomena as cell proliferation, viability, and cytotoxicity. When added to viable cells, blue, non-fluorescent resazurin undergoes a conversion to a highly fluorescent derivative. Both resazurin and its derivative are water soluble and have low toxicity, making it simple to handle and eliminating the washing, fixing, and extraction steps required for other commonly used cell proliferation assays.
Application: Cell viability assay
- Add resazurin solution (Product No. R0195) at a volume equal to 10% of the cell culture medium volume.
- Return cells to the incubator and continue the incubation for 2-24 hours.
- Measure the fluorescence intensity using 540-570 nm excitation and 590 nm emission wavelengths.
*Absorbance measurement may also be used in place of fluorescence measurement to estimate cell number – use a 570nm filter.
- 1) Bioluminescence of cellular ATP: a new method for evaluating cytotoxic agents in vitro
- 2) The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity