(1) Don’t trypsinize cells for too long when collecting them.
(2) Rotation speed should be no more than 1500 rpm during centrifugation.
(3) Before fixation with ethanol, cells should be resuspended into single cell suspension with a small amount of 1×PBS.
(4) Pipetting the final cell suspension through a nylon monofilament mesh screen with Ф44 micrometer openings immediately prior to analysis on the flow cytometer is highly recommended to remove large multi-cellular aggregates common in ethanol-fixed preparations, so as to ensure cells flow through the cytometer in a single line.
Through only cell culturing once, and one transient cell treatment, experimental materials to identify cell apoptosis with different instruments can be obtained, allowing simultaneous detection of morphology, biochemistry, cell cycle and DNA content. Unity of quantitative analysis and qualitative analysis can also be realized, which not only shortens the experiment time, reducing working time from 9 to 4 days, but also gets relatively satisfactory results.
Known methods to identify cellular apoptosis are characteristic of detection of targets of morphology, biochemistry and DNA content etc. Fluorescence microscopy needs cells to be seeded on coverslips and culturing until monolayers form. Its main shortcomings lie in the fussy steps in culturing cells on coverslips and the inferior picture quality resulting from cell-cell junction. As the most classical and dependable method to identify cell apoptosis, it is only qualitative and not easy to quantify the ratio of apoptotic cells. In classical electrophoresis detection，DNA is extracted with phenol-chloroform-isopentanol or phenol-chloroform and then precipitated with cold ethanol and sodium acetate after lysis buffer treatment, which is time-consuming and needs many centrifugation steps. Furthermore, phenol and chloroform are toxic, and too much centrifugation can shear DNA so as to decrease the experimental accuracy. Although pre-embedding cells with low-melting-point agarose and then performing pulse alternative field gel electrophoresis can avoid shearing DNA, time and expenditure on experiments will increase, moreover all these can only belong to qualitative analysis, at most semi-quantitative. Flow cytometric analysis, however, is simple, rapid, quantitative and multi-parametric.
Three independent experiments consume too much time, and experimental materials are not from the same group, so it is very hard to ensure the identity and veracity of results of former and latter experiments. Herein, the cells are prefixed in 70% cold ethanol, DNA is extracted with 0.2 M phosphate-citrate buffer at pH 7.8, then centrifuged at 1000rpm for 5~10 min so as to extract relative low-molecular-weight DNA into the buffer thoroughly, and then the supernatant is sequentially treated with RNase A and proteinase K and then subjected to electrophoresis directly. The cell pellets are dyed with propidium iodide, and a little amount is used for slide preparation and observation under a fluorescence microscope. The remaining cells are for flow cytometer analysis. Such one-station operation makes simultaneous detection of three targets come true. The advantages of this procedure are as follows:(1) The cells prefixed with 70% cold ethanol may be stored for several weeks or more before analysis without any significant DNA degradation. Moreover, treatment with ethanol also inactivates several pathogens and prevents cells from autolysis, thereby increasing the safety of sample handling; (2) This procedure permits the electrophoresis analysis of DNA extracted from the very same cell population that is subjected to measurements by flow cytometer to estimate DNA ploidy, the cell cycle distribution of non-apoptotic cells, the percentage of apoptotic cells, or other parameters. In other words, cells cultured once can be used to detect multi-targets only through a single transient treatment, which can shorten experimental time；(3) It simplifies the fussy steps of culturing cells on coverslips when morphological characteristics are detected under fluorescence microscope; (4) The procedure to extract relative low-molecular-weight DNA is simple and rapid, and uses no phenol, chloroform or other toxic reagents so as to reduce DNA damage by phenol- chloroform and the inconvenience of pulse field gel electrophoresis; (5) Treatment with 0.2 M phosphate-citrate buffer at pH 7.8 makes the degraded, low-molecular-weight DNA fragments from apoptotic cells ooze out of the cells completely, increasing the sensitivity and specificity of flow cytometer analysis; (6)The procedure can ensure the identity of experimental materials, enhancing the consistency and reliability of experimental results. In short, the procedure successfully uses the very same cell population to analyze the degraded DNA ladder of apoptotic cells, observe the morphological characteristics of apoptotic cells and determine the percentage of apoptotic cells. This is just as “one stone hits three birds”. As a sort of improvement on classical methods, researchers can select this procedure according to their different purposes and respective conditions.
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[5] David L. S., Robert D.G., Leslie A. L., Cells: A Laboratory Manual, Volume 1: Culture and Biochemical Analysis of Cells, (Patricia Barker, ed), Cold Spring Harbor Laboratory Press, New York， pp.15.1-15.12 (1998)
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[7] Cui Y.Y.，Xie H., Wang J.F. Transient detection of apoptosis of human liver cancer cells induced by Pinus massoniana bark extract (PMBE) in vitro, Chin. J. Histochem. Cytochem. 14, 80-83 (2005)
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