How to measure the number of living cells in a cell factory

A cell factory is a one-layer or multi-layer structure of cell culture consumables. During the cell culture process, we need to grasp the growth state of the cells in time before proceeding to the next step. The number of viable cells can be detected by trypan blue staining. The specific operation method is as follows:

1. For adherent cells, aspirate the medium, add the digestion solution, and digest in the incubator for an appropriate time. After the cells became round, the digestion was terminated by adding an equal or greater volume of medium than the volume of the digestate. After centrifugation, the cell suspension is made with culture medium;

2. Take 0.5ml trypan blue solution, 0.3ml PBS and 0.2ml cell suspension and mix well. Then the mixture was left for 1-2min. Use a capillary pipette or a 10ul small pipette tip to draw a small amount of the mixture and inject it into the hemocytometer;

3. Count at least 1000 cells and count the number of stained cells;

4. Wash the counting plate and coverslip with ultrapure water and 75% ethanol;

5. Normal cells will not be stained, only after the cells die, the cell membrane permeability becomes larger, trypan blue enters the cells, and the cells appear blue. The mixture should not be placed for too long, otherwise the normal living cells will also take up the dye, which will affect the counting accuracy;

6. Cell viability (%) = (total number of cells - number of stained cells)/total number of cells*100;

7. Like the cell counting method, trypan blue staining has a large error. It can only detect the early apoptosis level, and cannot show the difference between the control group and the experimental group.

In the process of culturing cells in a cell factory, there are always some cells that die due to various reasons. Mastering the number of living cells and cell viability is a key operation in cell culture experiments and an effective way to ensure the progress of the experiment.