Dead cells estimation

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... 

The clonogenic assay is normally exploited to analyze the effect of compounds on the symmetry of division in NSCs/CSCs. In this assay, the number of secondary spheres generated from the dissociation of a single sphere reflects the frequency of NSC/CSCs present in the original primary clone. This analysis also returns an estimate of the relative frequencies between symmetric proliferative (two SCs generated at each cycle) and symmetric differentiative (two differentiated and/or dead cells generated after cell division). 

Cell sorting takes place at a rate of 300,000 cells/min and for this reason a FACS machine is more usually used as an analytical tool rather than in a preparative mode. In addition to its use in cell cycle analysis, it can be used (a) to analyse the distribution of lymphocytes carrying a series of different surface antigens (e.g. to determine the proportion of T4 lymphocytes in a blood sample) (b) to estimate the proportion of dead cells in a population (i.e. cells which stain with propidium iodide without prior fixation) or (c) to determine the proportion of transformed cells (i.e. cells bearing a particular surface antigen) in a culture or biopsy (Watson, 1987). 

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 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. 

Lactate dehydrogenase is a useful marker because it is released upon cell death and is stable over short periods of time (5% loss per day) (Wagner et al, 1992), so that the concentration of LDH in the medium provides an estimate of the total number of (intact plus lysed) dead cells. Equations 4.2.1-4.2.5 are still valid, but a balance needs to be included for the effective cell concentration which is equal to the total cell concentration plus the concentration of cells that have lysed (i.e. what the total cell concentration would be if no cells had lysed) ... 

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 %.)... 

The use of adenylate kinase (AK) rather than ATP for biomass estimations has been advocated/ AK is a very stable enzyme and the assay does not differentiate between living and dead cells. However, AK may be used in hygiene control. The ATP level in ADP, i.e. the AK substrate, sets the detection limit of the assay. This is a problem since ADP easily disproportionate to ATP and AMP. 

The measurements of biomass and cell number described cannot, by themselves, discriminate between living and dead cells, and are therefore made in conjunction with a procedure for estimating the viability of the cells in the sample. 

Fig. 7. The effect of the cell cycle on the coupling of NAD catabolic events in K-562 cells. 15 x 10 cells were placed in 75 ml fresh medium and cultured without any further medium change for 1-6 days. Dead cells were removed by gradient centrifugation and the indicated parameters estimated (for details see Results and Discussion). Day 1 values were (ADP-ribosyl) transferase in permeabilized cells (11.8 pmol TCA precipitable NAD/1 x 10 cells/30 min) (13) NAD glycohydrolase as NAM generated extra-cellularly (185 pmol/1 x 10 cells/30 min), uptake of [ H]adenine labeled NAD (64 pmol/1 X 10 cells/30 min) (Figure 1) DNA synthesis (140,000 cpm pH]dThd/pg DNA/3 hr, 22 CiAnmol).

Another difficulty with the direct microscope count is the fact that the method views both viable and nonviable (dead) cells. Depending on stage in the growth cycle, as well as history of the sample, the ratio of viable to nonviable cells may vary considerably and makes comparing results to those of direct plating difficult. Because of this, plating normally provides lower estimates of viable populations than microscopy. To make the distinction between viable and nonviable cells, various stains and dyes can be used either singly or in combination (Section 14.4.2, 14.4.3, and 14.4.4). 

The result shown in the above example is a power density that has been exceeded in many studies, and thus this calculation provides good evidence that cells form more than a monolayer on a complete flat surface. For two layers of cells, we could simply double this number, and for three layers, triple it, and so forth. Cells cannot completely fill a surface, and dead cells may occupy some part of the surface, which would decrease the above estimate. More importantly, it must be recognized that most electrode surfaces are not completely flat and thus it is possible that we have underestimated the surface area and thus the maximum cell density. The result shown is slightly different from that obtained by Liu and Logan (2004) (2.2 W/m ) due to their use of a different yield (Y = 3.6 X lO cell/mol-Ac), smaller cells, and the lack of inclusion of the Coulombic efficiency in the calculation versus Ce = 0.84 used here. 

Other vital stains take advantage of different cellular properties which can be correlated with cellular physiology Propidium Iodide, Ethidium Bromide, Ethidium Monoazide, Calcofluor White have been widely used to indicate the presence of dead eukaryotes or prokaryotes cells. 2-(p-iodophenyl-)3)(p-nitro-phenyl)-5-phenyl tetrazolium chloride (INT) belongs to a class of stains which can be used to determine if a cell or hyphal compartments [180] can maintain an internal reducing environment (Fig. 20a). There are, however, still a large debate about the reliability of those techniques, depending upon the cells under consideration [181]. Calcofluor (Aex = 380 nm, Aem 420 nm) is a specific cell wall stain which enables to counts buds scars on Saccharomyces cerevisiae [29] to estimate the age of a cell. 

Organic PP is associated with detrital matter from dead and decomposing bacteria, phytoplankton, zooplankton cells, and periphyton, as well as from vascular plants, and organic phosphorus associated with particulate matter is termed as POP. POP is estimated as follows ... 

---