Harry A. Crissman

Cell-Cycle Analysis in 20 Minutes

Mithramycin added to mammalian cells fixed in aqueous ethanol is bound to DNA and fluoresces in direct proportion to the cellular DNA content. Quantitative fluorescence measurement by means of a high-speed flow system instrument provides a rapid method for cell-cycle analysis and for the first time permits continuous monitoring of cell-cycle kinetics during ongoing experiments.

Automated single-cell manipulation and sorting by light trapping

Following the recently reported trapping of biological particles by finely focused laser beams, we report on the automated micromanipulation of cells and other microscopic particles by purely optical means as well as on a newly observed interaction between particles in the trapping beam. A simple instrument is described which allows single cells to be positioned with high accuracy, transported over several millimeters, and automatically sorted on the basis of their optical properties. These operations are performed inside a small enclosed chamber without mechanical contact or significant fluid flow. Potential applications of this technique in experimental cell biology are discussed.

Chapter 12 Methods and Applications of Flow Systems for Analysis and Sorting of Mammalian Cells1

Publisher Summary This chapter discusses the methods and applications of flow systems for analysis and sorting of mammalian cells. Flow systems offer several advantages when compared with the various static systems which analyze cells on slides. In most flow instruments, each cell is exposed to the light beam for only a few microseconds; thus, problems with fluorescence decay are minimized. The successful application of flow systems for rapid, single-cell analysis is critically dependent upon preparative techniques which maintain the cells in a monodispersed state during fixation, staining, and measurement. In instances where fluorescent staining techniques are employed, the quality and specificity of cellular staining must also be evaluated to ascertain the reliability of analytical results. Automated analytical systems that are designed to perform rapid and precise measurement of individual cells cannot be totally relied upon to distinguish fluorescent cellular debris or cell clumps from properly stained single cells. Therefore, sample preparation involving both the disaggregation of tissue into single-cell entities and cell staining plays an extremely important role in flow-system methodology.

RAPID STAINING METHODS FOR ANALYSIS OF DEOXYRIBONUCLEIC ACID AND PROTEIN IN MAMMALIAN CELLS

Quantitative fluorescent staining and analysis of cellular deoxyribonucleic acid (DNA) were accomplished using three groups of reagents having different mechanisms of action for DNA binding. These reagents included (a) the fluorescent antitumor antibiotics mithramycin, chromomycin A3 and olivomycin; (b) the Feulgen reagents acriflavine and flavophosphine N and (c) the intercalating dyes ethidium bromide and propidium iodide. Propidium iodide (PI) was used in combination with fluorescein isothiocyanate (FITC) to stain both cellular DNA and protein, respectively. Multiparameter analysis of PI/FITC-stained cells provided a direct correlation of DNA and protein for cells in all stages of the cell cycle. Nuclear-to-cytoplasmic ratio determinations were also performed on PI/FITC-stained cells by analysis of the time duration of the red (DNA) and green (protein) fluorescence signals from each cell. These staining and analysis techniques provide alternative methods for directly determining the quantitative relationship between cellular DNA and protein and will be extremely useful in investigations where fluctuations of these parameters are of importance for assessing experimental results.

Cell cycle-dependent protein expression of mammalian homologs of yeast DNA double-strand break repair genes Rad51 and Rad52

Recently, human and rodent homologs of yeast repair genes Rad51 and Rad52 have been identified and proposed to play roles in DNA double-strand break (DSB) repair. In this study, cell cycle-dependent expression of human and rodent RAD51 and RAD52 proteins was monitored using two approaches. First, flow cytometric measurements of DNA content and immunofluorescence were used to determine the phase-specific levels of RAD51 and RAD52 protein expression in irradiated and control populations. The expression of both proteins was lowest in G0/G1, increased in S and reached a maximum in G2/M. No difference was found in the whole-cell level of RAD51 or RAD52 protein expression between gamma-irradiated and control cell populations. Second, cell cycle-dependent protein expression was confirmed by Western analysis of populations synchronized in G0, G1 and G2 phases. Analysis of V3, a hamster equivalent of SCID, indicates that the protein level increases of RAD51 and RAD52 from G0 to G1/S/G2 do not require DNA-PK.

Preparation of large quantities of synchronized mammalian cells in late G1 in the pre-DNA replicative phase of the cell cycle.

Abstract Chinese hamster cells, line CHO, can be synchronized in late G1 by growth in isoleucine-deficient medium for 30 h followed by resuspension in fresh, complete medium containing either hydroxyurea (to 10−3 M) or cytosine arabinoside (to 5 μg/ml) for 10 h. Cells which are then washed and resuspended in fresh, complete medium without drugs initiate DNA synthesis shortly thereafter, then commence dividing within 7 h. The technique yields large quantities of cells suitable for biochemical studies of events associated with initiation of genome replication and completion of interphase.

Cytometry of the cell cycle: Cycling through history

PREHISTORY: THE PAINS OF THE PRE–FLOW CYTOMETRY ERA For young investigators who presently enter their scientific pursuit focused on cell cycle and have flow cytometry (FCM) at their disposal, it may be difficult to visualize the hardship of experimental procedures during the precytometry era. Autoradiography (1) was then the predominant method for cell cycle studies. It was a cumbersome and time-consuming methodology. The radioisotope-labeled cells deposited on microscope slides had to be fixed and covered with photographic emulsion in nearly total darkness. This was particularly tricky when using the “stripping film” approach, and required the preparer to either be on a carrot diet or to consume large quantities of vitamin A to enhance his or her night vision. After careful air-drying, the autoradiographs had to be left in light-proof boxes for several days’ and sometimes weeks’ exposure. Then, again in the dark, the autoradiographs had to be processed through developer, rinse, and fixer, followed by drying. Subsequently, the cells had to be counterstained through the emulsion (which also was tricky, because the emulsion had a tendency to detach, ruining the specimen) and mounted under a coverslip. Quantitative analysis of autoradiographs was painful as well. One had to identify labeled cells located below the silver grains of emulsion by microscopy, and score by eye the labeling index (LI) by counting hundreds of cells per each sample. Sometimes it was necessary to count individual silver grains, to estimate the intensity of cell labeling. Such analysis could take a long time, leaving the investigator with painful eyes and phantom images of the silver grains residing in his or her retina for hours. Attempts to develop semiautomatic or automatic screening of autoradiographs based on backward light scattering by silver grains of emulsion were generally unsuccessful (1). Despite the hardship, important discoveries were made, and numerous autoradiographic techniques, designed to assess the cell cycle and kinetics of cell proliferation, were developed. In fact, the evidence that DNA replication is discontinuous during the cycle, occurring within the distinct time interval during the interphase, was obtained by autoradiography (2,3). It was observed that the radioisotope (P or H)-labeled DNA precursor thymidine was incorporated into nuclei by a fraction of the interphase cells only, leaving many cells with unlabeled nuclei. This finding provided the foundation for subdivision of the cell cycle into four major phases: preDNA synthetic interphase or postmitotic gap (G1), DNA synthesis phase (S), postsynthetic interphase or premitotic gap (G2), and mitosis (M). Perhaps the most elegant technique to measure kinetics of cell progression through the cycle was based on pulse-labeling cells with H-thymidine, followed by analysis of the percentage of labeled mitotic cells (3,4). Analysis of the kinetics of progression of the cohort of cells labeled during the short pulse in S phase through the narrow time-window of the M phase provided an accurate estimate of the duration of each phase of the cycle and of the whole cell cycle (Tc) (4). In vitro and in vivo applications of H-thymidine autoradiography yielded a wealth of information about cell cycle and the kinetics of cell proliferation of several normal and cancer cell models (4–6). Microspectrophotometry and microfluorometry were also applied in studies of the cell cycle, as techniques complementary to autoradiography. They were used to measure the content of DNA, RNA, and protein in individual cells. However, only few laboratories could afford such instruments, which were then generally homemade. Their development and maintenance required a significant investment and close collaboration of biologists with optical and mechanic engineers. During 1967–1968, one of us (ZD) had an opportunity to use these instruments at the Institute for Medical Cell Research and Genetics at the Karolinska Institute in Stockholm. Directed by Torbjorn Caspersson, the “grandfather” of cytometry (7), this laboratory had the most advanced microspectrophotometers and microfluorometers at the time, and was the Mecca for scientists from all over the world who were seeking a possibility to quantify DNA, RNA, or total protein in individual cells. Reservations to use the microspectrophotometer had to be made weeks ahead, as there was a long waiting line of investigators eager to measure cells. After some experience was gained, approximately 40 cells could be measured per hour. This number was then considered be adequate for statistical analyses in most publications. Needless to say, the cell analysis had to be biased

Chapter 13 Staining of DNA in Live and Fixed Cells

Publisher Summary Quantitative fluorescent staining and flow cytometric (FCM) analysis of cellular DNA remain the most rapid and reliable approach for assessing relative DNA contents of various cell types, as well as for obtaining cell-cycle frequency distributions and chromosome profiles of cell populations. Early studies comparing computer-fit analysis of DNA content histograms to data obtained by conventional tritiated thymidine labeling and autoradiography confirmed the accuracy of the FCM technique for cell-cycle analysis. In recent years flow cytometry has been used to elucidate other physiological parameters, which, in addition to DNA metabolism, regulate and control cell proliferation. Many current studies involve labeling and measuring cellular constituents such as proteins and RNA simultaneously with DNA. Cellular levels of such descriptors and others are known to be important indicators of cell-cycle progression capacity, cell growth, and function. The sample preparation method, the fluorochrome probe combinations, and the flow instrument capabilities for analysis of the various cellular descriptors will determine the choice of DNA labeling procedure.

Synchronization of human diploid fibroblasts at multiple stages of the cell cycle

Because of the scarcity of techniques for synchronizing the growth of cultured human diploid fibroblasts at multiple stages within the cell cycle, efforts were expended in this report to establish a set of protocols that would permit synchronization of cells at several different points throughout the cycle. The protocols that were developed to synchronize the growth of HSF-24 and HSF-55 cells, human foreskin-derived fibroblast cultures, were modifications of procedures employed to synchronize the growth of cultured rodent cells. Optimization of synchrony induction was directed by consideration of both the biochemical properties of the synchronized populations (determined via three-parameter flow cytometric measurements of DNA, RNA, and protein contents) and their kinetic behavior following reversal of the synchronization-inducing blockade (determined via combined flow cytometric analysis of DNA content, [3H]thymidine autoradiography, and measurement of increase in cell number). The conditions judged to yield the best results for studying events associated with production of a G0 block or for maintaining cells for prolonged periods in G0 were those in which the cells were grown to confluency in D-MEM supplemented with 10% fetal bovine serum. Procedures producing the best results for studying processes associated with the G0 to G1 transition, G1 events, and operations accompanying the transition from G1 to S, employed subconfluent growth for 48 h in alpha-MEM + 0.1% fetal bovine serum (alpha-MEM0.1F) followed by resuspension in alpha-MEM containing 10% fetal bovine serum (alpha-MEM10F). When the goal was to obtain cells in which to study very early S-phase events, satisfactory results were achieved by combining a 48-h period of subconfluent growth in alpha-MEM0.1F, followed by treatment for 24 h in alpha-MEM10F containing 5 micrograms/ml aphidicolin. For study of events occurring in mid- to late-cycle, acceptable results were achieved by combining a 48-h block in alpha-MEM0.1F with resuspension for 24 h in alpha-MEM10F containing 10(-3) M hydroxyurea followed by resuspension in drug-free alpha-MEM10F. The best results were obtained with these latter synchronization procedures (i.e., low-serum/high-serum + APC or HU/high serum) when the fetal calf serum was replaced with heat-inactivated calf serum. The success achieved in synchronizing the growth of these human diploid fibroblasts compared favorably/exceeded the results obtained with synchronized cultures of Chinese hamster ovary cells.

Changes in cell nuclei during S phase : progressive chromatin condensation and altered expression of the proliferation-associated nuclear proteins Ki-67, cyclin (PCNA), p105, and p34

Using multiparameter flow cytometry we have measured the nuclear DNA content of exponentially growing HL-60 cells in conjunction with protein content, nuclear forward light scatter, DNA in situ sensitivity to denaturation, DNA accessibility to 7-aminoactinomycin D (7-AMD), and content of the proliferation-associated proteins: cyclin (PCNA), p105, p34, and Ki-67. Multivariate analysis of the data made it possible to correlate changes in each parameter with the degree of cell advancement through S phase (amount of replicated DNA). A decrease of the protein/DNA ratio, lowered DNA accessibility to 7-AMD, increased sensitivity of DNA to denaturation, and increased ability of isolated nuclei to scatter light all paralleled cell progression through S phase. These changes indicate that during S phase chromatin progressively condenses and suggest that the condensation is associated with the efflux of nonhistone proteins from the nucleus. The increase in the content of the antigen detected by the Ki-67 antibody was observed to exceed the increase in DNA content during S phase and the rate of the Ki-67/DNA increase was higher during the second half of S phase. Thus, this protein appears to be primarily synthesized during S, especially late in S phase, and is degraded in G1. The ratio of cyclin (PCNA)/DNA remained rather constant whereas the contents of p105 and p34 proteins, when expressed per unit of DNA, both decreased during S phase. The data indicate that significant changes in structure and composition of chromatin take place during S phase and suggest that the composition of chromatin associated with the nonreplicated DNA is different compared to chromatin associated with the newly replicated DNA.