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