What is the principle of trypan blue exclusion test?
It is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue, Eosin, or propidium, whereas dead cells do not. In this test, a cell suspension is simply mixed with dye and then visually examined to determine whether cells take up or exclude dye.
What is the problem with trypan blue staining?
The staining of cells with trypan blue may rapidly increase the number of negatively charged residues on cytoplasmic proteins (8,19), which attracted more positively charged ions such as sodium, leading to high water influx and ruptured the already fragile cell membrane and cytoskeleton (3,20-22).
Why is trypan blue referred to as an exclusion dye?
Trypan blue is an azo dye. It is a direct dye for cotton textiles. Since live cells are excluded from staining, this staining method is also described as a dye exclusion method.
How accurate is trypan blue?
Over the years, there has been an understanding that trypan blue is inaccurate for cell viability under 80% without scientific support. We previously showed that trypan blue can alter the morphology of dead cells to a diffuse shape, which can lead to over-estimation of viability.
How do you dilute trypan blue?
Mix 1 part of 0.4% trypan blue and 1 part cell suspension (dilution of cells). Allow mixture to incubate appoximately 3 minutes at room temperature. Note: Cells should be counted within 3-5 minutes of mixing with trypan blue, as longer incubation periods will lead to cell death and reduced viability counts.
Why do dead cells stain blue?
Because live cells have an intact cell membrane, trypan blue cannot penetrate the cell membrane of live cells and enter the cytoplasm. In a dead cell, trypan blue passes through the porous cell membrane and enters the cytoplasm. Under light microscopy analysis, only dead cells have a blue color.
What is trypan blue assay?
The trypan blue staining assay allows for a direct identification and enumeration of live (unstained) and dead (blue) cells in a given population. In this assay, a cell suspension is simply mixed with trypan blue and then visually examined to determine whether cells take up or exclude the dye.
How do you calculate the dilution factor for cell counting?
Dilution Factor = Total Volume (Volume of sample + Volume of diluting liquid) / Volume of sample. Total viable cells/Sample = Viable Cells/ml x The original volume of fluid from which the cell sample was removed.
How is the trypan blue exclusion test used?
The dye exclusion test is used to determine the number of viable cells present in a cell suspension. It is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue, eosin, or propidium, whereas dead cells do not.
What makes trypan blue a bluish color?
Trypan blue is a ~960 Daltons molecule that is cell membrane impermeable and therefore only enters cells with compromised membranes. Upon entry into the cell, trypan blue binds to intracellular proteins thereby rendering the cells a bluish color.
How is trypan blue used to measure cell viability?
Using Trypan Blue to Measure Cell Viability One of the earliest and most common methods for measuring cell viability is the trypan blue (TB) exclusion assay. Trypan blue is a ~960 Daltons molecule that is cell membrane impermeable and therefore only enters cells with compromised membranes.
How to test trypan blue in a hemacytometer?
Protocol. Prepare a 0.4% solution of trypan blue in buffered isotonic salt solution, pH 7.2 to 7.3 (i.e., phosphate-buffered saline). Add 0.1 mL of trypan blue stock solution to 0.1 mL of cells. Load a hemacytometer and examine immediately under a microscope at low magnification.