Trypan Blue Stain

Trypan Blue Stain

Assay-kits

Product code: ACMBS-63
Specifications: 1x20mL, 1x100mL (Box)
Intended use: Used to check the integrity of cell membranes to determine if cells survive
Store temperature: 5℃～30℃
Expiry date: 24 Months

More Details

PrincipleMain componentsSample requirementTest procedureResult interpretationMethod limitationPrecautions

Principle

Trypan blue is a cell-reactive dye, commonly used to test cell membrane integrity to determine if cells survive. When cell damage or death, trypan blue can penetrate the denatured cell membrane into the cells, make it blue. The living cells can prevent the dye into the cell, it is not colored. In order to identify dead cells and living cells. Strictly speaking, trypan blue staining detects the integrity of the cell membrane. It is generally accepted that the integrity of the cell membrane is lost and the cell is considered dead. This is called the dye exclusion test.

Main components

Reagent composition	Main ingredient
Trypan blue stain solution	Trypan blue

Sample requirement

Prepare a single cell suspension of appropriate concentration.

Test procedure

1. Preparation of single cell suspension:

Adherent cells were collected and digested cell with trypsin-EDTA. Collect suspended cells, direct blow and beat the bottom of the bottle to obtain a cell suspension. The collected cells were centrifuged at 1000 ～ 2000 rpm for 3 minutes, the supernatant was discarded, add 1ml PBS resuspend the cell pellet, dubbed the appropriate concentration of single cell suspension, spare.

2. The single cell suspension and trypan blue staining solution mixed well in a 9: 1 ratio.
3. Counting: Live cells and dead cells were counted respectively with a cell counting chamber, in three minutes.

①Place the counting-specific cover glass in the center of the cell counting chamber.

② Cells were aspirated with a glass siphon and the siphon was allowed to flow out of the well on the underside of the coverslip or counting chamber until the coverslip was filled with liquid.

③ Count the cells in large squares that have not been stained blue, that is, the number of living cells.

Result interpretation

Microscopically, the dead cells were stained blue, while the living cells were colorless and transparent. Statistics of cell viability: viable cell ratio (%) = total number of living cells / (total number of living cells + total number of dead cells x 100%).

Method limitation

Only use for observed the examination of morphological staining.

Precautions

1. Requires well dispersed cells in the cell suspension, otherwise affecting the accuracy of the count.
2. Sampling before counting, should be well mixed cell suspension, especially when sampling multiple times more should pay attention to each sampling should be mixed well in order to count accurately.
3. In operation, pay attention to the coverslip cannot have bubbles, also do not let the suspension flow into the side tank, or you have to re-count it.
4. Trypan blue staining solution with slight toxicity, please pay attention to safety precautions.
5. Staining time should not be too long, or living cells also will be partially stained, affecting the counting results.
6. Timely tighten the cap after each use to avoid volatile or deterioration, and affect test result.
7. This kit must be used by professionals and the interpretation of results.
8. Should carefully read the instruction manual before using. Can only be used before expiration date, and good personal hygiene protection.
9. After use, disposal of waste should comply with the hospital or the environmental protection department requirements.
10. The production date, production batch number and expiration date on the package.

References

1. Peng Bin, Wu Jingjing, Li Ye, Xu Qian, Tang Lin, Wang Baohe. Methodology of trypan blue exclusion assay in adherent cultured cells-Chinese Journal of Laser Biology. 2011, 20 (2).

2. Ye Rongrong, Shen Qingping, Qiao Guangyan. 3 kinds of commonly used clinical crown bridge fibroblasts in vitro cultured rat cytotoxicity comparison. Shanghai Stomatology. 2008. 17 (3).

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