Product Information

Product Description

Trypan Blue is an azo, hydrophilic, acidic blue dye that can penetrate the cell membranes of dead and dying cells and stain them blue. Live cells can exclude Trypan Blue due to the integrity of their cell membranes. Therefore, live cells (live cells are transparent and colorless) and dead cells can be distinguished by color changes. Based on this principle, this product is widely used in routine assessments of cell activity levels. Trypan Blue can also stain collagen and amyloid proteins. The complex formed by Trypan Blue binding to proteins can emit red fluorescence. In addition, Trypan Blue (appropriate concentration) is also often used to quench autofluorescence or other fluorescent signals on the cell surface.

This product is 0.4% (w/v) Trypan blue staining solution dissolved in physiological saline solution, supplied sterile, cell culture grade.

Transportation and storage methods

Transport with ice packs, store at 4℃, valid for 1 to 2 months.

Precautions

1) Before cell counting, cells need to be thoroughly washed with physiological salt buffer or serum-free culture medium, because cells have a very high affinity for serum proteins, and residual serum proteins will cause a darker staining background.

2) For your safety and health, please wear a lab coat and disposable gloves when operating.

3) This product is for scientific research purposes only!

How to use

      1) For suspension cells, collect the cells by centrifugation, wash , and then resuspend them in an appropriate buffer such as PBS or HBSS to make a single-cell suspension. For adherent cells, first digest the cells with trypsin, and then prepare a single-cell suspension according to the method for suspension cells.

      2) Take 0.5 mL of single-cell suspension and mix thoroughly with 0.5 mL of 0.4% trypan blue staining solution in a 1:1 ratio, and stain at room temperature for 3-10 minutes.

【Note 1】: Because trypan blue is cytotoxic, prolonged staining will cause some dead cells to be stained, interfering with counting.

[Note 2]: If the cell density is relatively high, take 0.2 mL of single-cell suspension, dilute it to 0.5 mL with appropriate buffer, and then stain it with 0.5 mL of 0.4% trypan blue staining solution.

      3) Take a small amount of the above stained cells and add them to the blood cell counting plate. Count them under a low-power microscope and calculate the stained cells (i.e. damaged cells and dead cells) and total cells respectively.

【Note 1】: When adding stained cells with a pipette, gently add liquid along the edge of the coverslip to completely cover the entire chamber. Do not add too much or too little liquid.

【Note 2】: The number of cells in a 1 mm2 ^grid is usually controlled at 20-50 cells. When the number of cells exceeds 200, the dilution factor needs to be readjusted.

      4) Calculate the cell number and percentage of live cells according to the following formula:

      a. Calculate the number of cells per mL: Cells/mL = average number of cells in the 1 mm2 grid × dilution factor × 104 ^{; [} Note]: The dilution factor at this time is: the dilution factor of the single cell suspension obtained in step 2 after mixing with the trypan blue staining solution

      b, Total cell number: Total cells = (Cells/mL) × the total volume of the initial single cell suspension

      c, Cell viability value: Viability (%) = (total number of cells - total number of stained cells) / total number of cells × 100

【Note】: Normally, for healthy logarithmic phase cultured cells, the living cells account for at least 95%.