Trypan Blue Cell Culture Tested

Trypan Blue
Cell Culture Tested Product Number T 6146 Store at Room Temperature
Product Description
Molecular Formula: C34H24N6NaO14S4
Molecular Weight: 891.8
CAS Number: 72-57-1
Melting Point: > 300 °C
λmax: 607 nm (methanol)
Synonyms: Direct Blue 14, Chlorazol blue 3B,
Benzo blue 3B, Dianil blue H3G, Congo blue 3B, Anphtnamine blue 3BX, Benzamine blue 3B,
Azidine blue 3B, Niagara blue 3B, Diamine Blue 3B
Trypan Blue is a blue acid dye that contains two azo chromophores. It has been used to dye and print cotton and leather, but does not have as strong an affinity for silk or wool.1
Trypan Blue has been used in vital staining of various tissues. It has been used to stain collagen and amyloid. It is also used in chlamydospore agar for fungi.2 Trypan blue will probably not penetrate the cell wall of plant cells grown in culture. However, it probably would penetrate the walls of protoplasts. Trypan Blue is a vital stain recommended for use in estimating the proportion of viable cells in a population.3 The reactivity of this dye is based on the fact that the chromophore is negatively charged and does not react with the cell unless the membrane is damaged. Staining facilitates the visualization of cell morphology. Live (viable) cells do not take up the dye and dead (non-viable) cells do. Trypan Blue has a greater affinity for serum proteins than for cellular protein. If the background is too dark, cells should be pelleted and resuspended in protein-free medium or salt solution prior to counting.
Precautions and Disclaimer
For Laboratory Use Only. Not for drug, household or other uses. Preparation Instructions
This product is soluble in water (10 mg/ml), yielding a very dark blue solution. It is also soluble in normal saline or balanced salt solutions.
Procedure
Reagents required:
Balanced salt solution (BSS) or physiological saline Trypan Blue solution [0.4% (w/v)] in BSS or normal saline
Hemocytometer (Product No. Z35,962-9)
Pasteur pipettes
Microscope (10x)
1. Prepare a cell suspension in BSS (Hank's
Balanced Salt Solution, Product No. H 2153) or
physiological saline.
2. Transfer 0.5 ml of 0.4% Trypan Blue solution to a
test tube. Add 0.3 ml of BSS or physiological
saline to 0.2 ml of the cell suspension (dilution
factor = 5) and mix thoroughly. Allow the cell
suspension-Trypan Blue mixture to stand at least
5 minutes, but no more than 15 minutes. If the
cells are exposed to Trypan Blue for extended
periods of time, viable cells, as well as non-viable cells, may begin to stain.
3. With the cover-slip in place, use a Pasteur pipette
or other suitable device to transfer a small amount of the Trypan Blue-cell suspension mixture to both chambers of the hemacytometer. Carefully touch
the edge of the cover-slip with the pipette tip and
allow each chamber to fill by capillary action. Do
not overfill or underfill the chambers.
4. Starting with chamber 1 of the hemacytometer,
count all the cells in the 1 mm center square and
four 1 mm corner squares (see Diagram I). Non-
viable cells will stain blue. Keep a separate count of viable and non-viable cells.
5. Count cells on top and left touching middle line of
the perimeter of each square. Do not count cells
touching the middle line at the bottom and right
sides (see Diagram II).
6. Repeat this procedure for chamber 2. If more than
10% of the cells appear clustered, repeat the
entire procedure. Disperse the cells by vigorous
pipetting of the original cell suspension and the
Trypan Blue-cell suspension mixture. If less than
200 or greater than 500 cells (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 the
counting procedure to ensure accuracy.
8. Cell Counts - Each square of the hemacytometer,
with cover-slip in place, represents a total volume of 0.1 mm3 or 10-4 cm3. This is the conversion
factor for the hemocytometer. Since 1 cm3 is
approximately 1 ml, the subsequent cell
concentration/ml (and total cell number) can be
determined using the following calculations:
Cells/ml = (average count /square) x (dilution factor) x 104 [chamber conversion factor]
Example: (45 cells) x (5) x (104) = 2.25 x 106 cells/ml Total Cells = (cells/ml) x (the original or predilution volume) Example: (2.25 x 106) x 10 ml = 2.25 x 107
Cell Viability (%) = (total viable ÷ total viable and nonviable) x 100
Total viable = viable cells/ml x original volume Example:
Average viable count/square = 37.5
Viable cells/ml = (37.5 x 5) x 104 = 1.875 x 106
Total viable = (1.875 x 106) x 10 ml = 1.875 x 107
Cell viability (%) =
(1.875 x 107) ÷ (2.25 x 107) x 100 = 83%. References
1. The Sigma-Aldrich Handbook of Stains, Dyes &
Indicators, Green, F.J., ed., Aldrich Chemical Co.
(Milwaukee, WI: 1990), p.721-722.
2. Conn's Biological Stains, 9th ed., Lillie, R. D.,
Williams and Wilkins (Baltimore, MD: 1977),
p. 158.
3. Phillips, H. J., and Terryberry, J. E., Counting
actively metabolizing tissue cultured cells. Exp.
Cell. Res., 13(2), 341-347 (1957).
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