The MTT Cell Proliferation Assay

The MTT Cell Proliferation Assay

INTRODUCTION

Measurement of cell viability and proliferation forms the basis for numerous in vitro assays of a cell population’s response to external factors. The reduction of tetrazolium salts is now widely accepted as a reliable way to examine cell proliferation. The yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) is reduced by metabolically active cells, in part by the action of dehydrogenase enzymes, to generate reducing equivalents such as NADH and NADPH. The resulting intracellular purple formazan can be solubilized and quantified by spectrophotometric means.

The MTT Cell Proliferation Assay measures the cell proliferation rate and conversely, when metabolic events lead to apoptosis or necrosis, the reduction in cell viability. The number of assay steps has been minimized as much as possible to expedite sample processing. The MTT Reagent yields low background absorbance values in the absence of cells. For each cell type the linear relationship between cell number and signal produced is established, thus allowing an accurate quantification of changes in the rate of cell proliferation.

Component Volume Storage

MTT Reagent 25 ml 4°C

Detergent Reagent 2 × 125 ml Room Temp. or 4°C

EQUIPMENT REQUIRED BUT NOT SUPPLIED

Microtiter plate reader with 650- and 570-nm filters

Inverted microscope

Multi-channel pipette

37°C incubator

Laminar flow hood

Microtiter plate (flat-bottomed)

Sterile tubes (5 ml)

Serological pipettes

Sterile pipette tips

BASIC PROTOCOL

If you are familiar with the procedure and know the cell count to use in your specific assay, you may follow this basic protocol.

1 、Plate cells at 1000 to 100,000 per well.

2 、Incubate for 6 to 24 hours

3 、Add 10 μl MTT Reagent

4 、Incubate for 2 to 4 hours until purple precipitate is visible.

5 、Add 100 μl Detergent Reagent.

6 、Leave at room temperature in the dark for 2 hours.

7、Record absorbance at 570 nm.

Step Action

1、DETERMINING OPTIMAL CELL COUNTS

Use the protocol below to determine the optimal cell count and incubation period for your cell line. This determination should only have to be done once for each cell type. The data will be used thereafter in your experimental system following the protocol above.

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