Activation of an endonuclease causes DNA fragmentation (see Chapter 3) and such DNA can be extracted from the cells following their fixation and permeabilization such that less DNA in apoptotic cells stains with any DNA
4: Analysis of ceil death by flow and laser-scanning-cytometry HL-60 CTRL HL-60 CP! 3h
Figure 4. The distinction between live, early apoptotic, and late apoptotic/necrotic cells by LSC after their staining with fluoresce inated annex in V and PI as described in Protocol 3. Apoptosis of HL60 cells was induced by their incubation with 0.15 [j.M of CPT for 3 h as described (16, 17), (A)The live, non-apoptotic cells neither stain with annexin V-FITC nor with PI, (B) Early apoptotic cells stain with fluorescein but exclude PI while (C) late apoptotic and necrotic cells stain with both dyes. The representative cells from the sectors A, B, or C of the contour map were relocated, viewed under blue light incident illumination, and their colour pictures were converted to grey scale.
fluorochrome (12, 13). The degree of DNA degradation varies depending on the stage of apoptosis. the cell type, and often the nature of the apoptosis-indueing agent. Hence, the extractability of DNA during the staining procedure also varies. It has been noted that a high molarity phosphate-citrate buffer enhances extraction of the fragmented DNA (36). This approach can be used to control the extent of DNA extraction from apoptotic cells to the desired level and to obtain their optimal separation by flow cytometry, as described in Protocol 4.
Protocol 4. Detection of fractional DNA content
• DNA staining solution: dissolve 200 ^g of PI in 10 ml of PBS and add 2 mg of DNase-free RNase A (boil RNase for 5 min if it is not DNase free). Prepare fresh staining solution before each use
• DMA extraction buffer: mix 192 ml of 0.2 M Na2HP04 with 8 ml of 0.1 M citric acid: pH 7.8
1. Fix the cells in suspension in 70% ethanol by adding 1 ml of cells suspended in PBS (1-5 x 10e cells) into 9 ml of 70% ethanol in a tube on ice. Cells can be stored in fixative at -20°C for several weeks.
Zbigniew Darzynkiewicz et al. Protocol 4. Continued
2. Centrifuge cells (200 g, 3 min), decant ethanol, suspend cells in 10 ml of PBS, and centrifuge (300 g, 5 min).
3. Suspend cells in 0.5 ml PBS, into which you may add 0.2-1.0 ml of the DNA extraction buffer.
4. Incubate at room temperature for 5 min, centrifuge.
5. Suspend the cell pellet in 1 ml of DNA staining solution.
6. Incubate the cells for 30 min at room temperature.
7. Analyse the cells by flow cytometry.
• use 488 nm laser line (or a mercury arc lamp with a BG12 filter) for excitation
• measure red fluorescence (>600 nm) and forward light scatter Alternative methods
Cellular DNA may be stained with other fluorochromes instead of PI, and other cell constituents may be counterstained in addition to DNA. The following is the procedure used to stain DNA with 4.6-diamidino-2-phenylindole (DAPI).
1. After step 4 above, suspend the cell pellet in 1 ml of a staining solution which contains DAPI (Molecular Probes) at a final concentration 1 (jLg/ml in PBS. Keep on ice for 20 min.
2. Analyse cells by flow cytometry.
• use excitation with UV light (e.g. 351 nm line from an argon ion laser, or mercury lamp with a UG1 filter)
• measure the blue fluorescence of DAPI in a band from 460 to 500 nm
To be analysed by LSC the cells should be fixed, rinsed with phosphate-citrate buffer, and stained in suspension, as described above, then placed on microscopic slides under a coverslip for measurement. Alternatively, the cells may be cytocentrifuged, smeared, or attached electrostatically to a microscope slide (see Section 2). To attach cells to a microscope slide by cytocentrifugation follow the steps as below:
1. Add 300 jxl of cell suspension in tissue culture medium (with serum) containing approximately 20000 cells into a cytospin (e.g. Shandon Scientific) chamber. Cytocentrifuge at 1000 r.p.m. (—110 g) for 6 min.
2. Without allowing the cytospun cells to dry completely, fix them by immersing the slides in a Coplin jar containing 70% ethanol. The cells can be stored in ethanol for several days. After fixation, rinse the slides in phosphate-citrate buffer, stain the cells with PI as described above for cell suspensions, and measure their fluorescence by LSC.
4: Analysis of cell death by flow and laser-scanning-cytometry (a) (b)
50 100 150
50 100 150
Figure 5. Detection of apoptotic cells with fractional DNA content based on cellular DNA content analysis. To induce apoptosis the cells were treated with DNA topoisomerase II inhibitor, fostriecin, as described (37). The cells were then fixed in 70% ethanol, stained with PI and their fluorescence was measured by flow cytometry (FACScan), as described in Protocol 4. The population of apoptotic cells is represented by the sub-G1 peak (Ap).
Apoptotic cells have a decreased PI (or DAPI) fluorescence and diminished forward light scatter compared with the cells in the main peak (Gl) (Figure 5). It should be emphasized that the degree of extraction of low MW DNA from apoptotic cells, and consequently the content of DNA remaining in the cell for flow cytometric analysis, may vary dramatically depending on the degree of DNA degradation (duration of apoptosis), the number of cell washings, the pH, and the molarity of the washing and staining buffers. Therefore, in Protocol 4, step 3, add less or no extraction buffer (e.g. 0-0.2 ml) if DNA degradation in apoptotic cells is extensive (late apoptosis), and more (up to 1.0 ml) if DNA is not markedly degraded (early apoptosis) and there are problems with separating apoptotic cells from Gl cells due to their overlap on DNA content frequency histograms.
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