Digestion staining solution and crystal violet staining cell nucleus counting method

Abstract

The invention belongs to the technical field of biochemistry, and particularly relates to a digestion staining solution and a crystal violet staining cell nucleus counting method. In order to solve the problems that pancreatin digestive juice digestion time is not easy to control in a sheet-shaped carrier adherent culture mode, and digested cells cannot be completely separated from a net-shaped space in the prior art, the invention provides the digestive staining solution which is a mixed solution of crystal violet, citric acid and triton X100. The crystal violet staining cell nucleus countingmethod comprises the following steps of: (1) mixing a carrier carrying cells with the digestion staining solution, and staining and digesting the mixture to obtain a staining solution; and (2) carrying out cell nucleus counting on the staining solution. The invention further provides application of triton X100 in digesting adherent cells in a cell nucleus counting process. According to the crystal violet staining cell nucleus counting method, the phenomena of cell damage and even death caused by overlong time in the digestion process can be reduced, so that the accuracy of cell counting is improved.

Description

technical field [0001] The invention relates to the technical field of biochemistry, and specifically relates to a digestion staining solution and a method for counting cell nuclei by crystal violet staining. Background technique [0002] At present, the large-scale cultivation of cells and viruses by mainstream vaccine manufacturers in the market generally adopts culture methods such as microcarrier bioreactors, sheet carrier bioreactors, spinner bottles or cell factories. In a fixed-bed bioreactor, the sheet-like carrier is fixed in a basket-shaped area, and the stirring paddle is placed in the middle cavity without direct contact with the cells. The rotation of the paddle makes the culture medium flow upward through the carrier loading area, and from the The middle cavity flows back to the bottom to form a loop, and oxygen ventilation and pH adjustment are all carried out in the middle cavity. The structure of fixed bed (sheet carrier) bioreactor culture is as follows: ...

AI Technical Summary

Problems solved by technology

In the prior art, trypsin digestion solution or similar recombinant digestion solution is usually used for cell digestion and counting. The disadvantage of using trypsin digestion or recombinant digestion solution for digestion is that the length of digestion is difficult to control, and if the time is too short, it cannot be completely digested. Long time will lead to cell damage or even death. For conventional cell suspension counting methods (such as capacitance method), cell death will affect the counting results

In addition, for cells attached to the reticular space (such as sheet-like carriers), cells cannot completely escape from the reticular space even after digestion due to the spatial structure

This further leads to inaccurate cell counting methods using trypsinized or reconstituted digests

[0005] It can be seen that for the counting of adherent cells in fixed bed bioreactors, the accuracy of technical results is affected by the defects of digestive fluid

Images