Trypan blue stock

Trypan Blue. Stock solution 1 % trypan blue in distilled water filter and store at 4°C. After 2 to 3 weeks precipitates will form which may greatly falsify the interpretation of dye-uptake into the cells. Working solution 9 volumes LC -i-1 volume trypan blue stock solution prepare just before the experiment and keep at 37°C. 

Remember that precipitates in the trypan blue stock solution will falsify the result, since the final concentration of trypan blue in your working solution will be considerably lower. 

Trypan blue stock solution To make 100 ml, dissolve 400 mg of trypan blue dye in 0.9% saline. Make up the volume to 100 ml, filter-sterilize, and store at 4°C. 

Trypan Blue (Sigma-Aldrich), stock solution of 5 mg/mL in MilliQ water filtered using a 0.22 pm membrane filter (Millipore). 

Trypan blue stain (0.2% w/v stock solution in phosphate-buffered saline [PBS]). 

To quench fluorescence from the complexes that are associated with cells but are not internalized into cells and to determine the percentage of cells that have internalized the transfection complexes, add trypan blue (TB) stock solution to the cell suspension for FACS analysis to a final TB concentration of 1 mg/ml, and analyze the cells again on a flow cytometer according to Subheading 3.8 steps 8 and 9. 

Trypan blue 0.4% stock concentration of trypan blue (Sigma, St. Louis, MO). 

To perform a cell count, a 1 20 dilution of the cell solution is needed. This is done by making a 1 10 dilution with complete media and then a 1 2 dilution with 0.4% stock concentration of trypan blue. 

The stain may fade with time but can be restored by immersion in a dilute NaOH solution. Methanol lost by evaporation of the stock solution can be replenished. Trypan Blue is a suspected carcinogen and all suitable precautions should be taken. In the writer s laboratory all paper towels and liquids containing the stain are collected in a pail and incinerated. It is available from Aldrich Chemicals, 1001 West Saint Paul Ave., Milwaukee, Wisconsin, 53233. Tel (414) 273-3850, Fax (414) 273-4979. 

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