Preparation of cells for analysis by LSC Detection of apoptotic cells based on changes in nuclear chromatin

Several assays of apoptosis by LSC require cell fixation. In these assays the cells are attached to microscope slides by standard methods that include smear films, tissue sections, 'touch' preparations from the tissues, or cyto-centrifuging cell suspensions. Cytocentrifugation is often preferred over 'touch' or smear preparations because it flattens the cells on the slides, so more morphological details are revealed.

LSC measurement of total nuclear or cellular fluorescence is done by integration of the light intensity of individual pixels over the area of nucleus and/or cytoplasm (25, 26). Thus, the intensity of individual pixels, as well as the fluorescence area (number of pixels), is being measured. The intensity of the maximal pixel within the measured area is also recorded. Because of the high degree of chromatin condensation in apoptotic cells, DNA stains with a greater intensity per unit of the projected nuclear area in these cells (hyper-chromasia). The maximal pixel value of the DNA-associated fluorescence measured in the chromatin of apoptotic cells, therefore, is greater than in the non-apoptotic nuclei (30). The situation is analogous to that of the chromatin of mitotic cells, which is also strongly condensed. Apoptotic cells, therefore, similarly to mitotic cells (31), can be identified by high values of the maximal pixel of DNA-associated fluorescence (Figure 1).

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Figure 1. Detection of apoptotic cells by LSC based on DNA hyperchromicity in condensed chromatin. Apoptosis was induced by treatment of HL60 cells with 0.15 jjlM camptothecin (CPT) for 3 or 4 h, as described (16, 17). The cells were stained with PI according to Protocol 1. In the untreated, control culture (CTRL) only mitotic cells (M) have a high value of red fluorescence maximal pixel. In the CPT-treated cultures, concomitant with a loss of S phase cells, the cells with high values of red maximal pixel become apparent. After relocation, when viewed by fluorescence microcopy, their morphology, as well as the morphology of the cells with fractional DNA content, was typical of apoptotic cells (shown in the four illustrations on the right).

Protocol 1. Identification of apoptotic cells by changes in nuclear chromatin

Reagents

• stock solution of propidium iodide (PI): dissolve 1 mg of PI (available from Molecular Probes, Inc., Eugene, OR) in 1 ml of distilled water. This solution can be stored at 0-4"C for weeks.

• prepare 1% formaldehyde solution in PBS. This solution should also be made fresh.

• prepare a working solution of PI by adding 200 (il of the stock solution of PI into 10 ml of PBS. Add, and dissolve in this solution, 2 mg RNase A (the final concentration of PI and RNase should be 20 (¿g/ml and 0.2 mg/ml, respectively). This solution should be made fresh.

Method

1. Deposit the cells on the microscope slide, preferably by cytocentri-fugatlon. To attach cells by cytocentrifugation add 300 |xl of cell suspension in tissue culture medium (with serum) containing approximately 20000 cells into a cytospin (e.g. Shandon Scientific, Pittsburgh, PA) chamber. Cytocentrifuge at 1000 r.p.m. (~110g) for 6 min.

2. Without allowing the cytospun cells to dry completely,fix them by immersing the slides in a Coplin jar containing 1% formaldehyde in PBS, on ice for 15 min.

3. Transfer the slides into Coplin jars containing 70% ethanol. The cells may be stored in ethanol for several days.

4. After fixation, rinse the slides in PBS for 5 min. Stain the cells with the working solution of PI for 20 min at room temperature. Mount the cells under a coverslip in the working solution of PI and seal the preparation with melted paraffin or nail polish. Measure the cellular red fluorescence (>600 nm; integrated fluorescence and maximal pixel) by LSC, illuminating the cells at 488 nm.

Identification of apoptotic cells by LSC based on the highest pixel value for DNA-associated fluorescence, as shown in Figure i, is done in conjunction with DNA content analysis. Thus, DNA ploidy and/or the cell cycle position of apoptotic and non-apoptotic cells, can be determined at the same time as the estimate of the apoptotic index. Furthermore, the staining procedure is simple and can be combined with analysis of other constituents of the cell when the latter are probed with fluorochromes of another colour. The disadvantage of this approach is that it cannot discriminate between mitotic and apoptotic cells. Also, early G1 (post-mitotic) cells may have high fluorescence intensity of the maximal pixel (31). The distinction between apoptotic and mitotic cells is critical during treatment with agents such as taxol or other mitotic blockers, i.e. when mitotic cells undergo apoptosis. However, visual examination of the cells, or analysis of other morphometric features, such as nuclear to cytoplasm ratio, nuclear or cell area, forward light scatter, etc., as offered by LSC, can be helpful in these instances.

Several cytometric methods designed to identify apoptotic cells, or to study molecular or metabolic events associated with apoptosis, probe the cells that have vital functions preserved and therefore cannot be fixed prior to analysis. Analysis of plasma membrane transport function (e.g. 9,32), detection of phos-phatidylserine on the plasma membrane (5, 10; see Chapter 7), or probing the mitochondrial metabolism (7, 8; see Chapter 8) all require unfixed cells. Suspensions of live cells in appropriately prepared reaction media are generally used when such analyses are done by flow cytometry. In the case of LSC, however, the measured cells have to be attached to a microscope slide to be relocated for morphological examination, additionally probed by another fluoro-chrome(s), or stained with a light-absorbing dye for analysis by light microscopy.

Two different approaches can be used to attach live cells to microscope slides. The first involves direct cell culturing on microscope slides or coverslips. Culture vessels are commercially available that have a microscope slide for the bottom of the chamber (e.g. 'Chamberslide', Nunc, Inc., Naperville, II). The cells growing in these chambers spread and attach to the slide surface. Chambers made on glass rather than plastic slides are preferred as the latter often have high autofluorescence. Alternatively, the cells can be grown on coverslips, which then can be inverted over shallow wells on the microscope slides for analysis by LSC. This mode of attaching cells to slides is, of course, applicable only to cells that normally grow on the surface of flasks, e.g. cells of epithelial or fibroblast lineage, macrophages/monocytes, etc. It should be stressed, however, that because the cells detach during late stages of apoptosis, many apoptotic cells may be selectively lost if the analysis is limited to the attached cells only.

Cells that grow in suspension can be attached to glass slides by electrostatic forces. This is done by incubating them on microscope slides in the absence of any serum or serum proteins (27-29, 33). A short (15-20 min) incubation of the cell suspension in PBS, at room temperature and 100% humidity, in shallow wells on the microscope slides, is generally adequate to attach the cells to the slide surface. The live cells thus attached can be subjected to surface immmunophenotyping (33), viability tests, or intracellular enzyme kinetics assays (27-29). Following fixation, e.g. in formaldehyde, the electrostatically attached cells become firmly attached by the fixative. When such preparations are restained, a large majority of the cells (>95%) is found to be still attached and can be relocated by LSC (33). However, as in the case of cell growth on glass, the late apoptotic cells have a tendency not to attach, or may detach after the initial attachment.

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