Trypan Blue Staining Lab Report

Method

• Methodology is performed through 3 successive days:

First Day: Trypan Blue cell viability assay:

Description

Trypan blue staining is a simple way to evaluate cell membrane integrity (and thus assume cell proliferation or death) but the method is not sensitive and cannot be adapted for high through put screening. Short 96 well assay: EACH condition should be done in triplicate or more.

Steps

1. Trypsinize one T-25 flask and add 5 ml of complete media to trypsinized cells. Centrifuge in a sterile 15 ml falcon tube at 500 rpm in the swinging bucked rotor (~400 x g) for 5 min.

2. Remove media and resuspend cells to 1.0 ml with complete media.

3. Dilute your cell sample in Trypan Blue dye of an acid azo exclusion medium by
Repeat this procedure for chamber 2; knowing that if greater than 10% of the cells appear clustered, repeat entire procedure making sure the cells are dispersed by vigorous pipetting in the original cell suspension as well as the Trypan Bluecell suspension mixture. If less than 200 or greater than 500 cells (i.e., 20-50 cells/square) are observed in the 10 squares, repeat the procedure adjusting to an appropriate dilution factor.

7. Withdraw a second sample and repeat count procedure to ensure accuracy.

8. CELL COUNTS _ Each square of the hemocytometer, with cover-slip in place, represents a total volume of 0.1 mm3 or 10-!4 cm3 . Since 1 cm3 is equivalent to approximately 1 ml, the subsequent cell concentration per ml (and the total number of cells) will be determined using the following calculations: Cell per ml = the average count per square X dilution factor X 104 (count 10 squares)

9. Total cells = cells per ml H the original volume of fluid from which cell sample was removed.

10. CELL VIABILITY (%) = total viable cells (unstained) ÷ total cells (stained and unstained) X100.

The Circle indicates the approximate area covered at 100x microscope magnification (10x ocular and 10x objective). Include cells on top and left touching middle line (O). Do not count cells touching middle line at bottom and right (ϕ). Count 4 corner squares and middle square in both chambers (one chamber represented
Yellow MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) is reduced to purple formazan in the mitochondria of living cells.

The absorbance of this colored solution can be quantified by measuring at a certain wavelength (usually between 500 and 600 nm) by a spectrophotometer. The absorption max is dependent on the solvent employed. This reduction takes place only when mitochondrial reductase enzymes are active, and therefore conversion can be directly related to the number of viable (living) cells.

When the amount of purple formazan produced by cells treated with an agent is compared with the amount of formazan produced by untreated control cells, the effectiveness of the agent in causing death of cells can be deduced, through the production of a dose-response curve.

Solutions of MTT solubilized in tissue culture media or balanced salt solutions, without phenol red, are yellowish in color. Mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring, yielding purple MTT formazan crystals which are insoluble in aqueous solutions. The crystals can be dissolved in acidified isopropanol. The resulting purple solution is spectrophotometrically measured. An increase in cell number results in an increase in the amount of MTT formazan formed and an increase in