Trypan blue exclusion cell count

Trypan Blue exclusion and viable cell counts Cell viability by MTT... 

Count viable cells by Trypan blue exclusion and seed cells at the appropriate density in culture medium in untreated tissue flasks. 

Count the cells by trypan blue exclusion test in a Biirker-Tiirk cell counting chamber and calculate the correct dilution (see below). 

C. Hepatocytes are counted in a hemocytometer and viability is determined using the trypan blue exclusion method. Viability is always > 90 % with a yield of 2-3 x 10s cells. Cells are resuspended in medium and diluted to a final concteration of 1 x 106 cells/ml. 

Remove 1 ml of cell suspension into a disposable culture tube for a cell count. Determine the cell concentration of the suspension by direct counting and monitor the viability using the Trypan blue exclusion test. 

A sample is taken for counting (the trypan blue exclusion technique can be used to identify the percentage of dead cells) (see Note 6). 

Maintain the cells in exponential growth by seeding at a density of 4 x 105-6 x 105 cells/mL, and count daily until a density of 2 x 106 cells/mL is reached. During this interval the cells are growing in a strict exponential fashion. In practice, the cells are subcultured and maintained with fresh medium containing 10% serum every 3 d. The cell viability should be maintained at approx 90-95%, as determined by the trypan blue exclusion method. 

Count the cell numbers and determine the viability by Trypan blue exclusion method. 

For statistical analysis, the greater the number of counts per culture the more accurate will be the statistics. The absolute minimum is triplicate values. Figure 2 shows the selective toxicity of EF13 for HIV-chronically infected cells over uninfected cells established using the Trypan blue exclusion test. 

Figure 35.8 Frequency of trypan-blue (TB) negative cells against the a values before subculture. The a values were evaluated by direct observation according to Eq. (35.1). The TB-stained cells were counted twice by trypan-blue exclusion test among about 100 cells. This figure was reproduced by permission of the Society for Biotechnology, Japan.

Resuspend the extracted PBMC in 1 mL additive-free RPMI. Count the cells using a hemacytometer after trypan blue exclusion to estimate the total number of extracted live cells. (Live cells do not incorporate trypan blue while dead cells do and thus, become blue. The percentage of cell viability is typically above 90 %.)... 

Yeh et al. [10] tested chrysophanic acid, emodin (5), physcion (6), emodin-8-0-(3- D-glucoside (7), physcion-8-O-P-Dglucoside (8), and trans-piceid (16) on HL-60 cells to determine their cytotoxic effects. The cells were exposed to known concentrations of the compounds for 72 hours and, using the trypan blue exclusion method, the viable cells were counted. As revealed in Table 2, the results indicate that emodin is the most cytotoxic compound tested. The observed potency is in the order emodin > physcion-8-0- 3-D- glucoside > fraras-piceid > physcion > emodin-8-0- 3-D-glucoside > chysophanic acid. 

Figure 26.4 Antiproliferative effect of I2 in human cell lines. After the indicated times, aliquots were removed and the cell number was determined by direct counting using trypan blue exclusion. Data are expressed as mean SD (n = 5) p < 0.05 vs. control cells. MCF-7, mammary cancer LNCaP, prostate cancer NIH-3T3, normal fibroblast.

Exposure of an immortalised rat type II alveolar epithelial cell line to 10 fM CdClj for 24 h caused a significant decrease in viability (Timblin et al. 1998). After 48 h of exposure to 1,5, or 10 /iM CdCU a dose-dependent decrease in viability was observed at dl concentrations as determined by trypan blue exclusion and cell counting. Significant increases in reduced and oxidised glutathione levels were observed at 24 and 48 h in rat lung epithelial cells exposed to CdC. Hart et al. (1999) observed a maximum level of apoptosis (5-fold higher than control) in cultures exposed for 48 h to 20/ M... 

Figure 1. Inhibition of geranylgeranoic acid-induced apoptotic cell death by a-tocophrol. Increasing concentrations (10 100 pM) of a-tocopherol were added to HuH-7 cell cultures with (closed circle) or without (open circle) 10 pM geranylgeranoic acid. After overnight incubation, the number of the viable cells was counted using a Trypan blue dye exclusion method. Means S.E. (n=3) are shown.

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. 

Cell concentration in suspension can be determined through an optical microscope employing a hemocytometer for manual cell counting, or in a semi-automatic way using an electronic particle counter (such as a Coulter counter), as described in detail by Freshney (2005). Through dye exclusion (such as trypan blue), it is possible to determine viable cell concentration, that is the number of cells in a known sample volume capable of proliferating in favorable culture conditions. 

Fig. 1. Analysis of cytotoxicity of ascorbate (AA) for HTLV-IIlB-infecled H9 cells, as determine by trypan blue dye exclusion. Each point is the mean of four cell counts.

Trypan blue solution 93595 Fluka Xex 488 nm kern 675 nm Trypan blue is a blue acid dye that contains two azo chromophores. It is a large, hydrophilic, tetrasulfonated dye. Trypan blue solution may be used in trypan blue-based cytotoxitiy and prohferation assays. It is a vital stain that is not absorbed by healthy viable cells. When cells are damaged or dead, trypan blue can enter the cell, allowing dead cells to be counted. When trypan blue binds to proteins the resulting complex emits red fluorescence. The method is sometimes referred to as the dye exclusion method. 

Check the cell viability using trypan blue dye exclusion and count the viable cell number using a hemocytometer. 

Lu et al. (1993) observed the interaction of DLC (obtained by IBAD) with human embryonic kidney (HEK-293) cells using a haemocytometer for cell counting and trypan blue dye exclusion to assess HEK-293 cell viability in DLC-coated P-35 dishes. According to Lu et al. (1993), HEK-293 cells grew well there was no delayed attachment to the DLC-coated dishes compared with the control, and both the cells growing in the DLC-coated and control dishes had a cell viability of 60% on the first day of incubation that increased to greater than 90% on the second day of incubation. 

Tumoral cell lines were tested in the usual manner. The cell lines were trypsinized, cells suspended and counted, then diluted to 10 and plated onto Corning 1 ml well plates. The plates were incubated overnight in a 00% humidity carbon dioxide incubator and the next day the Dulbecco s Modified Eagle Medium (DMEM) sucked off and substituted with DMEM containing various microgram quantities of the platinum-containing polymer. Inhibition was tested for by both visual observation and employing trypan blue (an exclusion stain). 

Figure 6. Summary on the cytotoxic effects of RST extract, tetrandrine, and fangchinoline on PMA-activated neutrophils. Cell viability was determined by counting the vital cell by trypan blue (0.4%) exclusion assay in the end of the...

The cytocidal activity was examined by the dye exclusion method of McLimans et al. (1975). A cell suspension was prepared immediately before the start of each experiment. The suspension was adjusted to contain 2X10 cells per ml. 0.5 ml of AaHI (80 pg/ml) were added to 4.5 ml of cell suspension. At 30-minute intervals thereafter 0.5 ml of the cell suspension was taken out from the tubes and mixed with 0.1 ml trypan blue (0.4%). Five minutes later, the viable and nonviable cells were counted under a microscope. 

Separation analysis at the outlet of the detector must respect three major conditions. The first is the cell integrity (i.e., the diagnosis of the particle). This can be operated on line, by means of classical photometric devices operated in the light-scattering mode (opacimetry) at 254 nm. Off-line methods, after fraction collection, are possible and recommended, by microscopy and granulometric analysis (Coulter counting). The second objective is to analyze cell viability. Off-line methods after fraction collection are equally possible. The blue trypan exclusion test, motility measurements, or specific enzymatic activities (esterase) can be performed on an aliquot of the collected fraction. 

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