User:Abdurahman Jewaro Kumbi Aruse/sandbox

Cell viability Cells may die during the culture process or during handling and propagation. If you are establishing a certain concentration of live cells to start a culture, or need a certain number of live cells for analysis, it is important to distinguish between live and dead cells. Cell counting is also useful for estimating growth rates. Since cells are usually cultured in the millions, the number of cells is first counted from a small volume and then extrapolated to the total cell volume. To achieve this, all cells are dissociated, pelleted and uniformly resuspended in an appropriate volume of medium. A small amount of cell suspension is mixed with 0.4% trypan blue in a 1:1 dilution in an Eppendorf tube. Trypan blue dye only infiltrates non-viable cells, which can therefore be excluded from subsequent quantification (Segeritz and Vallier, 2017). Abdurahman Jewaro Kumbi Aruse (talk) 05:47, 6 February 2024 (UTC) For this, 10 μl of trypan blue cell mixture is loaded into the hemacytometer. Using an inverted microscope, phase contrast and at least 10 x magnifications, count all cells in the outer four squares. Viable cells contain a darker "halo" while non-viable cells stain blue/black. To determine the total number of viable cells from all four squares, the number of cells found is divided by 4 (to determine the average number of cells per 1 mm2), multiplied by 104 (to obtain the number of cells per milliliter), and multiplied by 2 (to account for the trypan blue dilution factor) and multiplied by the total volume of cell suspension medium (fig 4). The percentage of viable cells can be determined by dividing the number of unstained cells by the total number of cells and multiplying the ratio by 100. A healthy cell culture is characterized by 80-95% cell viability (Bhatia et al., 2019).