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Application of hematoxylin in measuring cell multiplication activity and toxic effect of medicine to cells

A cell proliferation and hematoxylin technology is applied in the application field of hematoxylin in the determination of cell proliferation activity and the cytotoxic effect of drugs, which can solve the problems of complex and expensive equipment, non-specific adsorption, unstable results, etc. Good effect and stable test results

Inactive Publication Date: 2009-12-16
INST OF BASIC MEDICINE OF SAMS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] To observe cell proliferation activity and cytotoxicity, four types of methods are routinely used: 1. Isotope method, mainly including: 3 H-TdR incorporation method, 125 I-UdR and 51 Cr release method, although this type of method is sensitive and specific, has many disadvantages such as environmental pollution and the need for complex and expensive instruments and equipment.
2. Flow cytometry analysis also has the advantages of sensitivity and specificity, but it also requires expensive and complicated instruments and equipment, which is not easy to promote and apply at the grassroots level
3. Although Giemsa staining, crystal violet, trypan blue and other staining methods are simple and economical, they have disadvantages such as unstable results and serious non-specific adsorption.
In this study, when using MTT colorimetry and CCK-8 colorimetry to observe the toxic effect of the Chinese medicine extract Golden Buckwheat on tumor cells, it was found that both MTT and CCK-8 had different degrees of non-specific reactions with the Golden Buckwheat extract, resulting in the results The fluctuation range is large, and the dose-dependent relationship is not obvious
At present, there are no studies and reports on its application to determine the in vitro proliferation activity of adherent cell lines and the toxic effect of drugs on adherent cells

Method used

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  • Application of hematoxylin in measuring cell multiplication activity and toxic effect of medicine to cells
  • Application of hematoxylin in measuring cell multiplication activity and toxic effect of medicine to cells
  • Application of hematoxylin in measuring cell multiplication activity and toxic effect of medicine to cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] (1) Hematoxylin staining method to detect the proliferation activity of adherent cells, as follows:

[0036]Take 1640 complete medium and add it to a 96-well cell culture plate, 100μl / well. Huvec and M21 cells were digested with 0.25% trypsin, the supernatant was discarded after centrifugation, and the corresponding cell concentration was adjusted with complete medium. Then add 100μl of cell suspension to the corresponding wells of the above 96-well cell culture plate, mix well and dilute by a factor of 2, set 8 dilutions in sequence, 3 replicates for each dilution, and then each well is supplemented with complete medium To 200μl, and set a hole of 1640 medium for the microplate reader to adjust the zero. Place the 96-well cell culture plate at 37°C with a volume fraction of 5% CO 2 Incubate for 44 hours under saturated humidity. Take out the 96-well cell culture plate and discard the supernatant, use an 8-channel micropipette to immediately add 4% paraformaldehyde solution...

Embodiment 2

[0043] (1) Hematoxylin staining method to detect the toxicity of drugs on tumor cell lines

[0044] Take M21, SGC7901 and VX2 cells respectively, digest with 0.25% trypsin and centrifuge, discard the supernatant, adjust the corresponding cell concentration with complete medium, and add them to 96-well cell culture plate in turn, 100 μl / well. Place the 96-well plate at 37°C, 5% CO 2 Incubate overnight under saturated humidity to make it adhere to the wall. Dilute VCR, 5-Fu, CDDP and golden buckwheat extracts in sequence in 5 or 6 different concentrations, and add them to each corresponding well of a 96-well plate, 100μl / well, 3 multiple wells / concentration, and 6-9 wells without Drug cell control and 1 hole 1640 medium microplate reader to zero the hole, the total amount of liquid in each hole is 200μl. Place the 96-well cell culture plate at 37°C and 5% CO 2 Incubate for 44 hours under saturated humidity. Take out the 96-well cell culture plate and discard the supernatant, immedia...

Embodiment 3

[0048] Example 3: CCK-8 colorimetric method to detect the toxic effect of golden buckwheat extract on tumor cells:

[0049] Take M21 and SGC7901 cells respectively, digest and centrifuge with 0.25% trypsin, adjust the corresponding cell concentration with complete medium, and add them to 96-well cell culture plate in turn, 100 μl / well. Place the 96-well plate at 37°C, 5% CO 2 Incubate overnight under saturated humidity to make it adhere to the wall. Dilute the golden buckwheat extract in 6 different concentrations and add them to each corresponding well of a 96-well cell culture plate, 100μl / well, 3 multiple wells / concentration, and a non-drug cell control 6-well and 1-well 1640 medium microplate reader Zero adjustment hole, the total amount of liquid in each hole is 200μl. Place the 96-well cell culture plate at 37°C, 5% CO 2 Incubate in an incubator for 44 hours. Take out the 96-well cell culture plate, gently aspirate 100 μl of supernatant with a micropipette, and then add 10 μ...

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Abstract

The invention discloses application of hematoxylin in measuring the attached cell multiplication activity and the toxic effect of a medicine to attached cells. In the invention, hematoxylin is utilized to dye cells, and then the absorbency value under a specific wavelength is measured, so that corresponding data and conclusions are obtained. The results of measuring the cell multiplication activity of different time phases (12h, 24h, 48h and 72h) of attached cells, such as SGC7901, M21, Huvec and the like for many times by the inventor through adopting a hematoxylin dyeing method all indicate that the method not only has high specificity but also has better repeatability, and CV is less than 3%. The invention has the advantages of high sensitivity, stable detection result, wide application range, low cost and the like.

Description

Technical field [0001] The invention relates to the application of hematoxylin in measuring the proliferation activity of adherent cells and the toxic effect of drugs on adherent cells. Background technique [0002] With the deepening of the development of anti-tumor drugs, the anti-tumor effect of more and more traditional Chinese medicines has attracted worldwide attention. In vitro screening of such drugs or their extracts is also an important part of the drug development process. It is necessary to use a stable and reliable detection method to observe the inhibitory activity of drugs on the in vitro proliferation of different cell lines. [0003] To observe cell proliferation activity and cytotoxicity, four types of methods are routinely used: 1. Isotope methods, mainly including 3 H-TdR incorporation method, 125 I-UdR and 51 Although the Cr release method is sensitive and specific, it has many disadvantages such as environmental pollution and the need for complex and expensi...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12Q1/18
Inventor 张建华王郡甫陈红
Owner INST OF BASIC MEDICINE OF SAMS
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