Trypan blue cell stain

Leakage of cell contents (e.g., lactate dehydrogenase) and entry of extracellular dyes (e.g., Trypan blue, DNA stains such as TOTO-3)... 

Typically, the integrity of the cell membrane is evaluated by identifying the percentage of cells taking up a vital dye termed trypan blue. Cells that take up trypan blue have defective cell membranes and therefore external loading or contact with materials that cause cells to have faulty membranes are likely to cause cell injury and death. Cell cytotoxicity is evaluated by contacting a surface of a material or the extract of a material or implant with cells in culture. If more than five percent of the cell population stains with dye after contact with the material or an extract then it is considered cytotoxic. 

Twenty uL of the BAL is mixed with 20 uL of trypan blue, which stains the dead cells blue. Living cells are counted under a microscope using a Neubauer hemacytometer. 

Use 0.4% Trypan Blue to stain cells and count viable cells at seeding. Viability should be at least 95% at this stage. Culture cells in 50 mL media in Coming Spinner Flasks at 8% CO2, 37°C, in a humidified incubator (see Note 1). 

Cells in trypan blue viability stain were enumerated microscopically. The spleen cells were mixed with an equal number of SP/2.0 myeloma cells in the semi-log growth phase in RPMI medium. The cell mixture was centrifuged (200 x g) for 10 minutes and the cell pellet was suspended in 1 mL of polyethylene glycol (3 000 - 4 000 mol. wt. range) at 37 °C. The suspension was mixed continuously for one minute, followed by the addition of 1 mL of RPMI medium and another one minute of continuous mixing. An additional 9 mL of RPMI medium... 

Cell count staining method with Trypan Blue (see e.g. [68])... 

Fig. 15. Effects of turbulent shear stress level and exposure time on cell viability measured by trypan blue staining. Cells were sheared in a concentric cylinder viscometer [1]...

Isolated hepatocytes incubated with ionic iron rapidly undergo lipid peroxidation. Some studies have not shown a consequent decrease in viability (as indicated by uptake of trypan blue or release of enzymes). This is probably a result of short incubation times, as changes in viability lag behind increases in lipid peroxidation, and may not occur for more than 2 h after lipid peroxidation begins (Bacon and Britton, 1990). Recent studies have shown strong correlations between increased lipid peroxidation [production of thiobarbituric acid (TBA) reactants] and loss of cell viability (trypan blue staining) (Bacon and Britton, 1989). The significance of the lag between lipid peroxidation and decreases in cell viability is as yet uncertain. 

The hypothesis is supported by two additional observations (24). First, mean cell volume for P. brevis in the absence of aponin remained constant for 8 hours, but, in the presence of aponin, a notable increase was observed within an hour and continued for eight hours. Second, Trypan blue (Cl 23850) tests indicated increased cell permeability in the presence of aponin viable, motile cells were only slightly stained swollen cells and cell debris were highly stained. 

Trypan Blue exclusion (TB) Dead cells blue stained Cell membrane Yes3 [34]... 

Fig. 6.2. Caco-2 epithelial cell monolayers cultured with T. spiralis L1 larvae in (A) the absence or (B) presence of 1 mg ml 1 rat monoclonal, tyvelose-specific antibody 9D4 (McVay etal., 2000). Monolayers were fixed and stained with trypan blue as described in ManWarren etal. (1997). (A) Serpentine trails of nuclei in dead cells are evident, revealing the paths travelled by larvae. (B) Tyvelose-specific antibody has inhibited the migration of the larva such that it is encumbered in cell debris and has pulled up a large area of the monolayer, creating a plaque (P). Bar = 50 urn. Photomicrograph prepared by C. McVay, TTUHSC, Lubbock, Texas. 

MTX interferes with the growth of cancer cells by inhibiting the metabolism of folic acid. Drug efficacy was evaluated in vitro by MTT assay, as described above, and by Trypan Blue exclusion. Trypan Blue is a non-vital dye excluded by viable cells, but selectively staining dead cells. According to Figure 13.7, higher suppression of cell... 

As the buffer used in these studies [86, 158] is hypotonic to rat peritoneal mast cells, incubations of greater than 10 min result in significant cell swelling, a large increase in membrane permeability, as judged by trypan blue staining, and eventually cell lysis. This raises some concern as to the location of the site... 

The Trypan blue stain can be used to stain the dead cell, but is excluded from the live cells. This gives a good estimate of the viable cells. The reactivity of trypan blue is based on the fact that... 

To tag with secondary antibody-magnetic particle conjugate add 10 pL of anti PE-MACS microbeads antibody conjugate per 106 cells, incubate at 4°C for 15 min and wash twice with the PBS buffer. Set aside 1 x 106 cell aliquot for FACS analysis. Determine the total cell concentration, and population viability using a hemacytometer and Trypan blue stain. 

The seeding of carcinoma cells must be performed based on a live, healthy cell count. Dead cells (those that stained with Trypan blue) are not to be considered because they are expected to sediment in the high-density region. 

Metastasis 2. Medium for tumor cell culture, e.g., Minimal Essential Medium Eagle (MEM) (Gibco, Invitrogen) 3. 10X Trypsin/EDTA dilute to final concentration lx with PBS (Sigma-Aldrich) 4. Standardized tumor cell suspension, radiolabeled 5. Hank s balanced salt solution, Ca++ and Mg++ free (CMF-HBSS) (Sigma-Aldrich, H-2387) 6. Trypan blue stain 7. Mouse vice 8. Heat lamps... 

Perform a viable cell count using trypan blue stain exclusion and adjust the viable cell concentration to the desired number per milliliter with CMF-HBSS. 

FIGURE 8.10 Chronological sequence of images of a viability assay on a single Jurkat T cell, (a) Live cell perfused with trypan blue dye. Since the cell is alive, it is not stained, (b) Cell perfused with methanol, which causes cell membrane permeabilization, followed by cell death, (c) Permeabilized cell perfused with dye it is rapidly (< 5 s) stained. The entire assay is performed in less than 2 min [368]. Reprinted with permission from the American Chemical Society. 

Take some cell suspension and dilute with an equal volume of 0.1% trypan blue (stains dead cells Appendix 2). 

Viability - widely used to characterize animal cell cultures - is estimated according to the ability of individual cells to catalyze a biochemical reaction, e.g. the reduction of Methylene Blue or Trypan Blue to the respective leuko-form [65,332] after staining, blue cells are distinguished microscopically from noncolored cells. This method may give a significant overestimation of cell viability because lysed or lysing dead cells are not accounted for. Flow cytometric approaches are mentioned above (see Sect. 3.1). 

To verify the state of the membrane preparation after sonication, mix 1 vol of the membrane with 1 vol of trypan blue and examine by microscopy. If after 3 soni-cations live cells are still found, continue to sonicate. 80-90% of the cells should take up the trypan blue before proceeding to the next step. Trypan blue stains dead cells. 

The cells in the biological fluids are stained with trypan blue for determination of viability and with crystal violet containing citric acid (0.5 gm crystal violet and 10.5 grams citric acid in 500 mL dH20) for cell counts. 

To measure performance, to achieve reproducibility or to make comparative studies, a means of quantifying the cell population is needed. Classically, direct counts of cell numbers using a microscopic counting chamber (haemocytometer), usually in conjunction with a vital stain (e.g. Trypan blue) to distinguish viable and non-viable cells, is used. However, all vital stains are subjective and cannot give absolute values, and cell numbers take no account of differences in cell size/mass. The method is simple, quick and cheap, and requires only a small fraction of the total cells from a cell suspension. 

To ensure that a cell culture is growing exponentially it is useful to know the percentage viability and percentage of dead cells and hence the stage of growth of the cells. This can be estimated by their appearance under the microscope, because live healthy cells are usually round, retractile and relatively small in comparison to dead cells, which can appear larger, crenated and non-refractile when in suspension. The use of viability stains such as Trypan blue ensures a more quantitative analysis of the condition of the culture. Trypan blue is a stain that will only enter across the membranes of dead/non-viable cells. 

Monitor cell growth at least daily by taking a small sample from the side arm (remove flask to a tissue culture cabinet) and carrying out a cell count (Trypan blue stain and a haemocytometer). 

Assay for cell viability by staining an aliquot of the cells with trypan blue and counting the unstained viable cells, using a hemocytonrreter. 

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