Neutrophil activation monitored by flow cytometry: stimulation by phorbol diester is an all-or-none event.

The population dynamics of single-cell stimulation was analyzed by monitoring autofluorescence by flow cytometry. Stimulation of the respiratory burst in human neutrophils by 12-O-tetradecanoyl phorbol-13-acetate (TPA) caused a decline in highly fluorescent cells (characteristic of resting neutrophils) and a corresponding increase in the number of weakly fluorescent cells (characteristic of activated neutrophils). Increasing concentrations of TPA caused increasing numbers of cells to shift from the highly fluorescent population to the weakly fluorescent population without the appearance of intermediate populations. Thus the neutrophil respiratory burst, a component of neutrophil cytotoxic response, is triggered in an all-or none fashion.

High-speed chromosome sorting.

Dual-beam high-speed sorting has been developed to facilitate purification of chromosomes based on DNA staining with the fluorescent dyes Hoechst 33258 and chromomycin A3. Approximately 200 chromosomes per second of two types can be sorted from a suspension of chromosomes isolated from human lymphoblasts while fluorescent objects (chromosomes, debris fragments, chromosome clumps, and nuclei) are processed at the rate of about 20,000 per second. This sorting rate is approximately ten times that possible with conventional sorters. Chromosomes of a single type can be sorted with a purity of about 90 percent. DNA from the sorted chromosomes is suitable for construction of recombinant DNA libraries and for gene mapping.

Fluorescence-activated cell sorting analysis of the induction and expression of acute thermal tolerance within the cell cycle.

We have examined the cell cycle specificity of 45.5 degrees heat-induced toxicity and the induction and expression of thermal tolerance. Ultrapure populations of G1-, S-, and G2-M-phase cells were obtained through sequential centrifugal elutriation and flow cytometric cell sorting of Hoechst 33342-stained cells. We found no interaction of Hoechst 33342 with hyperthermia under staining conditions that gave good cytometric resolution of DNA distributions. Single dose-response survival curves indicated that S phase was the most sensitive to 45.5 degrees hyperthermia (Do = 1.97, 1.26, and 1.95 min for G1, S, and G2-M, respectively). Both S and G2-M phases exhibited a decreased ability from G1 to accumulate sublethal heat lesions as evidenced by decreased heat survival curve shoulders (Dq) = 13.7, 9.51, and 8.39 min for G1, S, and G2-M, respectively). Thermal tolerance, as measured by the decreased inactivation slope of the split-dose treatment, could be induced and expressed in G1, S, and G2-M phases. However, both the magnitude and temporal expression of tolerance were dependent on the position of the cell within the cell cycle at the time of the initial heat treatment. S-phase cells exhibited slightly less thermal tolerance as compared to G1 cells given isosurvival thermal induction doses as measured by the split-dose inactivation rate constants (heated/control = 8.37 and 5.62 for G1 cells at 12 and 24 hr and 7.68 and 5.27 for S-phase cells at 12 and 28 hr). Also, split-dose survival curves for cells heated in G2-M indicated a near total inability to accumulate heat-induced sublethal damage. Simultaneous bivariate (90 degrees light scatter and DNA content) progression analysis of heated replicates indicated that tolerance could probably be expressed in those cells which moved into other cycle compartments following the initial heat treatment. For instance, G1-phase cells preheated for 20 min began progression into normally heat-sensitive S phase between 24 and 28 hr after the heat treatment. This corresponded to approximately the time of maximal thermal tolerance expression. [3H]Thymidine suicide experiments also indicated that the ultimately clonogenic cells began movement into S phase at or near the time of maximal tolerance. In this case then, tolerance expression appeared to supersede the S-phase acute heat sensitivity. Heated S-phase cells began progression into G2-M between 4 and 12 hr, which corresponded temporally to large amounts of tolerance expression4 +

Effect of cell cycle position on the survival of 9L cells treated with nitrosoureas that alkylate, cross-link, and carbamoylate.

The relationship between cell cycle position and cytotoxicity was studied in 9L rat brain tumor cells treated with nitrosoureas that, depending on their structures, can alkylate or alkylate and cross-link DNA and/or carbamoylate biomolecules. Because pure populations of G1-, S-, and G2-M-phase cells could not be obtained with the centrifugal elutriation methods used, drug sensitivity of cells in each phase of the cell cycle was estimated using a mathematical model that accounts for variation in enrichment of elutriated fractions. 1,3-Bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea, 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea, which alkylate and cross-link DNA and carbamoylate biomolecules, and 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose (chlorozotocin), which alkylates and cross-links DNA but cannot carbamoylate biomolecules, killed more cells in G1 and G2-M phases than in S phase. N-Ethylnitrosourea, which alkylates and carbamoylates but does not form DNA interstrand cross-links, was more toxic to cells in S phase than in other phases. Cell kill caused by N,N'-bis(trans-4-hydroxycyclohexyl)-N-nitrosourea, a compound that carbamoylates only, increased progressively through the cell cycle from G1 to M. Nitrosoureas that cross-link DNA were more cytotoxic than nitrosoureas that do not cross-link DNA, although the latter had phase specificity. The results suggest that the increased sensitivity of G1- and G2-M-phase cells to chloroethylnitrosoureas is related to the formation of DNA interstrand cross-links.

Enrichment of murine hemopoietic clonogenic cells by multivariate analyses and sorting.

Multivariate analyses, dual beam flow cytometry and sorting, and list mode data processing were used to distinguish and enrich committed and pluripotent stem cells in mouse bone marrow. These cells were discriminated on the basis of their forward angle and perpendicular light scatter characteristics and their Hoechst 33342 fluorescence intensity. Myeloid committed progenitors (CFU-GM) and spleen colony-forming units (CFU-S) (day 9 and day 13) were enriched 100-fold by sorting on the basis of high forward angle light scatter, intermediate perpendicular light scatter, and very low HO fluorescence intensity. Approximately 10% of the sorted cells formed colonies in the CFU-GM assay and 2% formed CFU-S colonies. Morphologic analysis of the sorted subpopulation revealed 92% blast immature cell types. The DNA distribution of the sorted subpopulation, assessed by propidium iodide staining, indicated that 98% of the progenitor-enriched subpopulations contained 2N DNA content. This separation procedure offers a simple method to obtain preparations highly enriched in clonogenic cells in one pass through the cell sorter.

High resolution dual laser flow cytometry.

Flow cytometers based on optical sensing utilize external light sources and fluorescent dyes to measure one or more specific components or properties of individual cells or subcellular particles in liquid suspension. To provide for independent excitation of two dyes used in double staining experiments we have constructed a high resolution flow cytometer that uses two laser beams to provide two wavelengths of excitation. These beams are separated spatially so that cells flow through them sequentially, with a time separation of about 20 musec. Since the dyes are excited sequentially their emission occurs at different times and their emission spectra may overlap without causing any difficulty in analysis. We have developed new light collection optics that permit up to four measurements to be made on each cell. This approach greatly increases the number of dye combinations that can be used in flow cytometry, thus removing a significant limitation of single illumination instruments.

A study of acid phosphatase and dipeptidyl aminopeptidase II in monodispersed anterior pituitary cells using flow cytometry and electron microscopy.

A brief historical review of cytoenzymology is presented from the time of introduction into electron microscopy to the present, where the direction for quantification of an enzyme in single cells appears most promising by fluorescent staining. First attempts are reported to quantitate acid phosphatase (AcPase) and dipeptidyl aminopeptidase II (DAP-II) in monodispersed anterior pituitary cells from lactating and postlactating rats by flow cytometry, fluorescent, and electron microscopy. 3-Hydroxy-flavone is introduced as a new fluorescent cytochemical stain for AcPase, useful in flow cytometry but of only limited use in fluorescent microscopy. Histograms for AcPase indicate a single peak of cells staining more intensely in cell preparations from postlactating over lactating animals. Histograms for DAP-II staining indicate two distinct populations of cells present in the lactating and only one in the postlactating rat anterior pituitary gland. The application of dual laser staining indicates that not all cells stain for both enzymes. Electron microscopy shows the subcellular localization of DAP-II to be limited to lytic bodies and in mammotrophic cells to some secretion granules.

Investigations in high-precision sorting.

We have investigated the accuracy with which droplets containing cells can be sorted individually onto known and thus relocatable positions. The presence and random arrival of cells and particles in the sorter jet disturbs the orderly production and deflection of droplets, causing a dispersion of sorted droplet trajectories. The magnitude of this dispersion is a function of the phase relationship between the arrival of a cell at the end of the jet and the droplet formation. Using a modified Becton Dickinson Fluorescence-Activated Cell Sorter, we selected for sorting only those droplets that formed with a cell near the center of the droplet. We used this technique to sort Lewis lung tumor cells. The dispersion of droplet positions was reduced from over 3% to about 1% of an average deflection of typically 15 mm for a nozzle with a 50-micron diameter orifice. Sorting onto a surface such as magnetic tape or a microscope slide introduces another uncertainty in position because the cell may be located anywhere within the wetted radius of the droplet on the slide. Sorting onto less-wettable surfaces reduces the wetted radius and thus the variation in cell position.

Flow cytometry of human gynecologic specimens using log chromomycin A3 fluorescence and log 90 degrees light scatter.

Flow cytometry and electronic cell sorting are being investigated to screen gynecologic specimens for cervical neoplasia. Cellular DNA content is quantitated by Chromomycin A3 fluorescence and cell size is quantitated by 90 degrees light scatter; the logarithms of the measured intensities are used to produce a two parameter histogram. To determine the cell types responsible for signals in various histogram regions, systematic electronic cell sorting is performed. The sorted fractions are sedimented into microscope slides and stained by the Papanicolaou technique. The cells in each fraction are identified by conventional cytomorphologic criteria. Morphologic analysis of sorted cells reveals histogram regions corresponding to specific cell types. One very important region contains the highest concentration of signals from abnormal cells and is therefore the best region to analyze for specimen abnormality. However, because a significant number of signals in this region are from normal cells, specimens cannot be diagnosed by their analysis. Another important histogram region is composed primarily of signals from endocervical columnar and metaplastic cells. The presence of such cells is a good criterion for specimen adequacy, therefore analysis of signals in this region is essential to assess specimen adequacy for automatic screening.

Measurement of mammalian sperm deoxyribonucleic acid by flow cytometry. Problems and approaches.

Measurement of mammalian sperm deoxyribonucleic acid content is of importance in several areas of biomedical research. When measured in flow systems with orthogonal axes of illumination, flow and detection, an unexpected, distorted distribution consisting of a narrow peak with a lateral extension to the right is observed. Several lines of evidence lead to the conclusion that this effect is an optical-geometric artifact attributable to the flat shape and high index of refraction of mammalian sperm heads. This artifact disappears when an epiillumination flow system is used in which the optic axes for illumination and detection and the flow axis are all coincident. Other approaches also eliminate the artifact. The resulting coefficients of variation observed after acriflavine-Feulgen staining are 4-5%, short of the goal of 1.5% required to distinguish between human sperm bearing X and Y chromosomes and to develop a mutagen test system using mice.