Trypan blue solution preparation

P 82] Dilution-type mixing was accomplished with the fluorescent dyes acridine orange (0.01% solution in 20 mM in TE buffer see below) or trypan blue (prepared in 0.85% saline) contacted with buffer solution (TE buffer 10 mmol f4 Tris-HCl, pH 7.4, 1 mmol 1 1 EDTA, pH 8.0) [164]. Images were taken by a laser scanning confocal microscope. Profiling data analysis was employed along detection lines. 

The Trypan dyes are bisazo compounds distantly related to the sulfonamides. Trypan blue, a bluish-gray, water-soluble powder, was one of the first drugs to be used for the treatment of piroplasmo-sis and trypanosomiasis. Trypan blue is effective against B. caballi but not B. equi. In horses, a freshly prepared 1-2% solution of Trypan blue can be administered by slow i.v. injection at 2-3 mg/kg for premunition. Rapid i.v. administration of Trypan blue may result in shock. Subcutaneous administration of Trypan blue should be avoided because it causes skin sloughing. Trypan blue stains the mucous membranes and other tissues blue and is, therefore, not used commonly. 

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. 

The dose-dependence of pore-forming activity for SLO is rather steep. Therefore prepare a series of 1 1 dilutions of SLO in LC. As outlined below, wash the cells with KRH, add 50 xl of your SLO dilutions to the wells for 7 min, aspirate and add trypan blue working solution. Leave the solution for 10 min (for reproducibility, the time of incubation with SLO and with trypan blue is important). Check the degree of permeabilization under the light microscope. The correct dilution of SLO is characterized by 100% of blue cells and minimal loss of cells. 

The effects of sodium chlorite on membrane components and antioxidant depletion have been studied in rabbit corneal epithelial cells, human conjunctival epithelial cells, phospholipid vesicles prepared from egg yolk and GSH in solution. Incubation of phospholipid vesicles with 3.5 mmol sodium chlorite/l for up to 2 h had no effect, whereas incubation for 48 h resulted in lipid depletion and an increase in lipid oxidation. Sodium chlorite was found to be a very potent GSH oxidizing agent at a GSH/sodium chlorite ratio of 0.5, GSH was depleted after 5 min. GSH depletion was also seen in rabbit corneal epithelial cells and human conjunctival epithelial cells incubated with 3.5 mmol sodium chlorite/l or 0.55 mmol sodium chlorite/l. At 3.5 mmol/l, sodium chlorite caused rapid loss of cell viability in the corneal cells, as assessed by trypan blue staining and loss of adherence. At 0.55 mmol/l, sodium chlorite had very little effect over the first few hours but decreased viability after 24 h. The conjunctival cells appeared to be less sensitive than the corneal cells. No oxidatively modified lipids could be detected in the cells following sodium chlorite treatment, and no effects were seen in levels of cytosolic antioxidants (Ingram et al., 2003). 

A stain solution is made by dissolving 0.2 gram Trypan Blue in 25 mL me anol. Prepare a 30% solution of caustic soda (30 grams NaOH in 70 mL distilled water) and add it to the stain solution in a dark plastic bottle. 

Prepare the samples for counting by mixing 10 pL of the cell suspension from Subheading 3.3.1, step 7 with 10 pL of trypan blue. Use 10 pL of this solution to manually or automatically count the cells see Note 21). Cell viability at this stage is an important quality control checkpoint. If the cells do not have a viability of 90 % or more, we do not proceed with screening ( eeNote 22). 

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