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A counting method and cell nucleus technology, applied in the field of biochemistry, can solve the problems of poor accuracy, cell damage, death, etc., and achieve the effect of reducing cell counting errors
Pending Publication Date: 2021-02-23
成都柏奥特克生物科技股份有限公司
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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 deat
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Embodiment 1
[0032] The formula of the digestive staining solution used in this embodiment is as follows:
[0033] Crystal violet 1.00g / L;
[0034] Citric acid 21.00g / L;
[0035] Triton X-100 1.00ml / L.
[0036] The specific process of cell counting is as follows:
[0037] 1) Add an appropriate amount of water for injection in the water bath in advance, turn on the power, and set the temperature to 37°C.
[0038] 2) After the temperature of the water bath reaches 37°C, place the crystal violet staining solution in the water bath and preheat for 30 minutes.
[0039] 3) Sampling 10 sheet carriers after cell culture, put them in a 50ml centrifuge tube, add 10ml of the above digestion and staining solution, shake well, put in a water bath, 37°C water bath for 30min, take it out and shake vigorously every 10min.
[0040] 4) After step 3) is completed, take 1ml of the staining solution in the centrifuge tube and add 9ml of water for 10-fold dilution, and shake well.
[0041] 5) Take the dilu...
Embodiment 2
[0058] The test process of the present embodiment is the same as that of Example 1, the difference being that the digestive staining liquid formula used in the present embodiment is as follows:
[0059] Crystal violet 1.50g / L;
[0060] Citric acid 20.00g / L;
[0061] Triton X-100 1.50ml / L.
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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: ...
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