Forward Angle Light Scatter Research Articles

Assessment of DNA content and cell cycle distribution of erythroid and myeloid cells from bone marrow.

A method is described for the measurement of DNA index and cell cycle distribution in purified erythroid and myeloid populations from human bone marrow. Erythroid cells were prepared after complement mediated lysis of non-erythroid marrow cells. Myeloid cells were obtained by fluorescence activated cell sorting by forward and wide angle light scatter. Mononuclear marrow cells were prepared with a density gradient. Nuclei prepared from the separated populations were stained with propidium iodide. Myeloid cells had a higher DNA index than erythroid cells, and the mononuclear preparation had an intermediate value. There were more erythroid than myeloid cells in the S and G2M phases of the cell cycle. These lineage differences are particularly relevant when considering data derived from unseparated bone marrow cells, and further experiments are needed to determine the origin of these anomalies.

Transforming growth factor-beta-induced growth inhibition and cellular hypertrophy in cultured vascular smooth muscle cells.

We have explored the hypothesis that hypertrophy of vascular smooth muscle cells may be regulated, in part, by growth inhibitory factors that alter the pattern of the growth response to serum mitogens by characterizing the effects of the potent growth inhibitor, transforming growth factor-beta (TGF-beta), on both hyperplastic and hypertrophic growth of cultured rat aortic smooth muscle cells. TGF-beta inhibited serum-induced proliferation of rat aortic smooth muscle cells (ED50 = 2 pM); this is consistent with previously reported observations in bovine aortic smooth muscle cells (Assoian et al. 1982. J. Biol. Chem. 258:7155-7160). Growth inhibition was due in part to a greater than twofold increase in the cell cycle transit time in cells that continued to proliferate in the presence of TGF-beta. TGF-beta concurrently induced cellular hypertrophy as assessed by flow cytometric analysis of cellular protein content (47% increase) and forward angle light scatter (32-50% increase), an index of cell size. In addition to being time and concentration dependent, this hypertrophy was reversible. Simultaneous flow cytometric evaluation of forward angle light scatter and cellular DNA content demonstrated that TGF-beta-induced hypertrophy was not dependent on withdrawal of cells from the cell cycle nor was it dependent on growth arrest of cells at a particular point in the cell cycle in that both cycling cells in the G2 phase of the cell cycle and those in G1 were hypertrophied with respect to the corresponding cells in vehicle-treated controls. Chronic treatment with TGF-beta (100 pM, 9 d) was associated with accumulation of cells in the G2 phase of the cell cycle in the virtual absence of cells in S phase, whereas subsequent removal of TGF-beta from these cultures was associated with the appearance of a significant fraction of cycling cells with greater than 4c DNA content, consistent with development of tetraploidy. Results of these studies support a role for TGF-beta in the control of smooth muscle cell growth and suggest that at least one mechanism whereby hypertrophy and hyperploidy may occur in this, as well as other cell types, is by alterations in the response to serum mitogens by potent growth inhibitors such as TGF-beta.

Single–Cell Light Scatter as a Probe of Refractile Body Formation in Recombinant Escherichia Coli

Accumulation of foreign proteins as inclusion bodies in Escherichia coli was investigated using single–cell light scatter measurements from flow cytometry. Significant increases in forward–angle light scatter and right–angle light scatter were observed for E. coli strains overproducing two types of interferon and two types of veterinary growth hormone. The original host morphology before the induction of foreign gene expression was found to have a significant effect on the subsequent enhancement of light scatter by inclusion body formation. The light scatter distributions obtained by flow cytometry differed significantly from volume distributions obtained using Coulter sizing. Thus flow cytometry may provide information on cloned gene product accumulation at the single–cell level that is not available from other methods.

Flow Cytometric Analysis of Porcine Peripheral Blood Leukocytes Infected With Pseudorabies Virus

The susceptibility of fractionated porcine peripheral blood leukocytes (PBL) to pseudorabies virus (PRV) was studied by flow cytometry and defined by viral antigen expression. Viral antigens on the surface of infected cells and cell viability were evaluated by forward angle light scatter (FALS), 90-degree light scatter (90LS), green fluorescence (FITC-anti-PRV), and red fluorescence (propidium iodide). Approximately 10% of infected mononuclear cells from healthy pigs expressed cell-surface PRV antigen. Cell-surface fluorescence and cell type were confirmed by sorting live positive cells for microscopy. In sorted positive samples, the lymphocyte versus monocyte ratio was approximately 50%:50%, defined by morphology. Positive lymphocytes represent 5.75% of total mononuclear cells. When cells were stimulated with phytohemagglutinin (PHA) and lipopolysaccharide (LPS) before infection, mitogen-stimulated T-lymphoblasts showed increased susceptibility to PRV (40.7% positive) and died of infection. Monocytes, particularly adherent monocytes, were highly susceptible (40% to 71.4% positive). Granulocytes appeared to be refractory. The relative susceptibility of various PBL populations was compared by normalizing lymphocyte susceptibility to 1 as follows: resting total lymphocytes (1); B-lymphocytes (0.67); T-lymphoblasts (7.08); total monocytes (4.27); adherent cells (4.03 to 10.88); adherent monocytes (6.95 to 12.42); granulocytes (0.24). These findings suggest a possible mechanism by which PRV could have an immunosuppressive effect as well as a pathway for dissemination of PRV.

Acousto-optic laser-scanning cytometer.

An instrument has been developed that uses a computer-controlled rapidly scanning laser beam to make cytometric measurements on cells or particles and which can measure low levels of fluorescence when using low-power lasers (Gershman, Hoffman, and O'Connell, "Methods and Apparatus for Analysis of Particles and Cells.") The method used is based upon acousto-optic principles of light diffraction. A vertically polarized 5-mW He-Ne laser is directed into an acousto-optic Bragg cell in which a portion of the incident light undergoes a small angular variation or deflection. Suitable optics focus the beam to a 25 microns diameter spot, at the 1/e2 point, in a sample cuvette while translating the angular variation into a linear scan. The cuvette enclosing the sample is slowly moved (approximately 1 micron/ms) via a stepper drive into the scanning beam while the forward angle light scatter sensor is monitored for the presence of valid signal events. When an event occurs, appropriate software optimizes the position of the focused laser beam onto the cell. Subsequently, scanning is stopped to allow for cell interrogation times that last for milliseconds or longer.

Characterization of granulosa cell subpopulations from avian preovulatory follicles by multiparameter flow cytometry.

The objective of the present study was to characterize the granulosa cell populations from individual hen (Gallus domesticus) preovulatory follicles at defined stages of follicular maturation using multiparameter flow cytometry. Granulosa cells were fixed and stained with three fluorochromes that selectively bind to DNA (Hoechst 33342, blue), RNA (pyronin Y, red), or protein (fluorescein isothiocyanate, green). A flow cytometer equipped with a three-laser excitation system was used to analyze three colors of fluorescence from stained cells. Forward angle light scatter and axial light loss measurements were made on each cell to determine relative cell size. In addition, the ratios of RNA to protein and DNA to protein were measured. The major findings obtained from correlated measurements of cell cycle (DNA), protein, RNA, cell size, and ratios were: 1) the percentage of proliferating cells decreased while cell size increased during follicular maturation; 2) two subpopulations of granulosa cells were identified within each follicle based on relatively high and low protein contents; the fraction of cells in the high protein subpopulation increased, and the fraction of cells in the low protein subpopulation decreased during follicular maturation; 3) the high and low protein subpopulations also differed in cell cycle distribution, RNA content, and cell size; and 4) the distribution of cells into the two subpopulations and the degree of proliferation were influenced by stage of the ovulatory cycle, primarily in the most mature follicles. The results demonstrate the dynamic heterogeneity of the granulosa cell populations from individual ovarian follicles and show the influences of follicular maturation and stage of the ovulatory cycle on cell growth and metabolism.

A predictive assay for human tumor cellular response to hyperthermia using dansyl lysine staining and flow cytometry

The heat response of five human tumor biopsies has been examined using the fluorescent probe dansyl lysine and multiparameter flow cytometry. Dansyl lysine has previously been shown to possess specificity for heat killed mammalian cells. The human tumors tested included a cervical squamous cell carcinoma, malignant melanoma, colon adenocarcinoma, ovarian carcinoma, and a mesothelioma. The samples were excised, mechanically disrupted into single cell suspensions and heated in vitro for various lengths of time at 45°C. The cells were returned to 37°C incubation for 12 to 15 hours prior to staining with dansyl lysine. The fraction of cells staining dansyl lysine was quantitated by flow cytometry after gating on high forward angle light scatter and 90°C light scatter. This gate excluded much of the normal cell contamination within the tumor sample. The data show that the heat response of human tumor biopsies varied significantly, with cervical carcinoma and malignant melanoma being the most resistant and the mesothelioma and ovarian carcinoma the most heat sensitive. Finally, evidence is presented for the expression of thermotolerance in ovarian carcinoma and mesothelioma biopsies pre-heated in vitro. Dansyl lysine appears to be useful in measuring the intrinsic cellular heat sensitivity of human tumors and in determining the kinetics of decay of thermotolerance following an initial heat exposure.

Limitations of flow cytometry for the specific detection of bacteria in mixed populations

Flow immunofluorescence (FIF) techniques were established for the specific detection of the bacteria Escherichia coli, Legionella pneumophila and Bacillus anthracis spores after staining with flourescein-conjugated antibacterial antibody. For each bacterial type, a comparison was made of gating on narrow forward angle (NFA) light scatter and on the red fluorescence (Red Flu) signal available from staining with the nucleic acid dye propidium iodide. No universal gating method was found, since Bacillus spores did not take up propidium iodide and only a part of the Legionella population gave detectable NFA scatter signals. The efficiency of detecting bacteria stained with antibody remained constant with differing concentrations of the specific bacterium, and the estimate of the count for specific bacteria expressed as a fraction of the total cytometer count fell sharply with bacterial concentration. This effect was apparently due to cytometer noise inherent in the high sensitivity of detection needed for particles as small as these bacteria. The noise did not originate in the photomultipliers and was evidently the result either of light scatter from sub-micron particles in the sheath fluid or scatter from optical components. Part of the noise could be removed by selective gating, but there remained a noise component overlapping with the NFA scatter and Red Flu signals from the heterologous bacteria, i.e., those not stained with specific antibody. In consequence, at the low bacterial concentrations used no meaningful cytometer count could be obtained for the excess of the unstained bacteria and the proportion of specific bacteria in the mixed population could not, therefore, be calculated.

Detergent treatment of dictyostelium discoideum cells allows examination of internal cell type‐specific antigens by flow cytometry

Monoclonal antibodies are used extensively in flow cytometry to identify subpopulations of cells differing in surface antigens. Conventional studies on living cells do not allow analysis of internal antigens, because antibody molecules do not pass through an intact plasma membrane. It is important for developmental studies on Dictyostelium discoideum that not only surface but also internal antigens be analysed. Here techniques are reported that make possible such studies by permeabilising cells with mild detergent treatments using digitonin. Flow cytometer profiles of unfixed cells show that antigens recognised by two monoclonal antibodies, MUD102 and MUD3, are found inside subpopulations of cells in the D. discoideum slug. Double-labelling experiments were carried out to demonstrate that the antigens recognised by these antibodies are present inside prespore but not prestalk cells. The detergent treatment leads to loss of forward-angle light scatter, but 90 degrees light scatter of cells is not greatly affected. While fixed cells sometimes gave satisfactory results, internal labelling did not reliably demonstrate the two subpopulations observed with unfixed cells.

Nonspectific cytotoxic cells in fish ( [formula omitted][formula omitted]) VI. Flow cytometric analysis

Nonspecific cytotoxic cells (NCC) from channel catfish ( Ictalurus punctatus ) were enriched using flow cytometry (EPICS R V, 753). Parameters of forward angle and 90° light scatter were utilized to sort cytolytically functional populations of NCC. Fullbright (GR. II) 9.75 μm diameter fluorescent polystyrene microspheres were used for optical alignment. Cells obtained from the anterior kidney, spleen and peripheral blood were analyzed by one and/or two parameters of forward angle light scatter (FALS) and log integral 90° light scatter (L90°LS). Populations identified from FALS or 90°LS histograms were sorted and cells were tested for cytotoxicity against NC-37 target cells. Anterior kidney contained at least four different sized populations of NCC. Spleen cells were sorted by FALS and four different populations of NCC detected. Peripheral blood contained little NCC activity. Enrichment of NCC activity from anterior kidney tissue was compared using Percoll R density gradient separation and flow cytometry. Three to four fold higher cytotoxic activity was obtained from sorted cells compared to the population obtained from 45.5% Percoll R. Additional experiments to determine the enrichment of sorted NCC were conducted by analyzing the number of enriched NCC required to kill an individual NC-37 target cell. Approximately 1.5 - 2.0 NCC could lyse a chromium-51 labelled target cell. These studies demonstrated that NCC could be enriched by size and granularity discrimination using flow cytometry. Effector:target cell ratios of 10:1 and lower produced significant lysis when sorted cells were analyzed.

The application of flow cytometry in the study of natural killer cell cytotoxicity

A flow cytometric assay was developed to detect the cytotoxic function of natural killer (NK) cells. This procedure employed a single fluorochrome and forward angle light scatter to differentiate dead cells from live cells. When results obtained by flow cytometry were compared with 51Cr release assay, this assay showed high specificity and sensitivity. When propidium iodide was used to stain target cells killed by NK cells, we found that NK cell cytotoxicity was not cell cycle specific.

Flow cytometric analysis of ploidy in the axolotl, Ambystoma mexicanum

AbstractAlthough recent experiments on pattern formation during amphibian limb regeneration have relied on the use of triploid marked cells, the purity of the triploid donor has not been proven. To test the purity of the donor, blood (which contains noncycling nucleated red blood cells) was collected from the gills of 12 pressure‐induced triploid axolotls from two unrelated spawnings. Blood was also collected from 12 untreated sibling animals to serve as diploid controls. The blood samples were placed into a propidium iodide stain solution that had been adjusted to amphibian tonicity by the addition of distilled water. Samples were analyzed on an EPICS V (Coulter) flow cytometer. A minimum of 20,000 cells were analyzed in each sample at a flow rate that did not exceed 1,000 cells/second. In the diploid group, 88.6% of the cells were collected in channels of the DNA histogram corresponding to a 2C G1/G0 DNA content (channel 71±10) and 0.9% were collected in channels corresponding to a 3C G1/G0 DNA content (channel 106±10). In the triploid group, 99.2% of the cells were collected in nondiploid channels and only 0.8% were collected in channel 71±10. Utilizing the average peak channel number for the diploid group of 12 animals an average haploid channel number of 35 was calculated. The triploid group of animals exhibited DNA ploidy that averaged one haploid channel number above that of the diploid group. Based on forward angle light scatter, nuclei obtained from triploid red blood cells were observed to be larger than nuclei from diploid blood. Preliminary data are presented which demonstrate that the ploidy characteristics of solid tissues (brain, cornea, spleen, muscle, and limb blastemal mesenchyme) agree with those presented for blood.

Identification and characterization of liver nonparenchymal cells by flow cytometry.

Our objective was to correlate biochemical or functional properties that could be measured by fluorescence to individual nonparenchymal cell types by the simultaneous detection of forward angle light scatter and fluorescence. Cells were released from liver following selective digestion of hepatocytes by Pronase perfusion. To fractionate the cells according to size, isolates were subjected to unit gravity sedimentation on a 1 to 5% sucrose gradient. Flow cytometry was used to analyze the resulting fractions as well as the original unfractionated cells. This reaffirmed that forward angle light scatter is linearly related to the sedimentation velocity (size) of the cells and that it can be used to assess cell sizes in an unfractionated population. Endocytosis of fluorescently tagged particulates was studied by injecting either fluorescein isothiocyanate-tagged colloidal albumin or Coumarin-tagged latex beads (0.57 micron diameter) prior to Pronase perfusion. Beads were found only in the largest cells (Kupffer cells). Since endothelial cells could endocytose only colloidal albumin, they were readily differentiated from nonendocytosing, nonsinusoidal cells ("lymphocytes"). Staining cell protein with fluorescein isothiocyanate made possible the determination of relative protein content per cell ("lymphocyte": endothelial:Kupffer cell was 0.57:1.0:2.0). To determine relative esterase activities, mixed cell isolates were incubated in fluorescein diacetate ("lymphocyte":endothelial:Kupffer cells was 0.13:1.0:2.4). Uptake of rhodamine 123 was used to assess mitochondrial function ("lymphocyte":endothelial:Kupffer cells was 0.45:1.0:14.6). Mitochondrial volume per cell is known to be 1:2 for endothelial:Kupffer cells. Therefore, the large difference in rhodamine uptake is not related to volume.

Flow cytometric analysis and sorting of live female rat anterior pituitary cell types by forward angle and perpendicular light scatter: effect of 17 beta-estradiol.

Administration of physiological concentrations of 17 beta-estradiol (E2) to ovariectomized rats resulted in changes in the forward angle light scatter (FALS) and perpendicular light scatter (PLS) signals of anterior pituitary lobe cells. One day after steroid treatment some cells showed increases in PLS signals to an intermediate ridge position lying between the two ridges seen for cells prepared from oil-treated ovariectomized rats. Over the first 5 days of E2 treatment, the major effect was a progressive increase in the FALS signal of cells in this intermediate ridge position. Increases in PLS signals continued over the 12-day treatment period. Since the results of a cell sorting experiment showed that PRL cells predominated in the intermediate scatter ridge, it was concluded that E2 increases both size (FALS) and secretory granule content (PLS) of mammotrophs. Cells in the low and high scatter regions were unaffected by E2. Those in the low scatter region were shown, by both electron microscopy and light microscopic immunochemistry, to be enriched in agranular, unstained endothelial-follicular cells. The high-scatter region contained predominantly GH cells. Administration of E2 to pituitary cells cultured in serum-free or serum-containing media for 1-12 days had no effect on the FALS-PLS bivariate distribution. However, the light scatter ridges produced from cells in culture were compressed into a single ridge of narrow PLS and broad FALS signal intensity with increasing time in culture.

Flow cytometric analysis and sorting of live male rat anterior pituitary cell types by forward angle and perpendicular light scatter.

Light scatter patterns produced by living cells in the flow cytometer are known to provide useful information with regard to their size and internal structure. The purpose of this study was to determine if light scatter signals produced by live male rat anterior pituitary cells could be used as markers to aid in the identification and separation of different hormone-containing cell types. The typical light scatter pattern (4 X 10(5) cells/sample X 15 min) had three ridges in the forward angle light scatter (FALS) perpendicular light scatter (PLS) bivariate cell distribution. FALS signals could be correlated with the size of different cell types and PLS signals with their content of cytoplasmic secretory granules. Agranular cells dominated the low PLS ridge while moderately granulated PRL cells and heavily granulated GH cells dominated the medium and high PLS ridges, respectively. These light scatter patterns were reproducible both within and between different cell suspensions. Inclusion of dopamine in the pituitary gland dissociation medium, a treatment known to increase intracellular PRL contents of mammotrophs, increased the intensity of the PLS signals of a large population of cells, presumably PRL cells. Pituitary cells prepared from different aged male rats also showed changes in light scatter. Cell sorting on the basis of FALS-PLS signals established the relationship between cell type and light scatter pattern.

Fluorescence-activated cell sorter analysis of bulk-isolated porcine oligodendrocytes

We describe here the methods by which enriched populations of oligodendrocytes were isolated from adult porcine brains using Percoll density gradient centrifugation and their immunological properties analyzed by a fluorescence-activated cell sorter (FACS). The analyses by immunofluorescence microscopy and FACS indicated 95% and 93% purity of oligodendrocytes in the cell preparations when galactocerebroside antibody, an oligodendrocyte-specific marker, was employed. Further FACS analysis indicated that 90% of the cells with the forward angle light scatter characteristics of oligodendrocytes were immunolabelled by the monoclonal antibody HNK-1. The FACS sorting capability was then used to correlate the forward angle light scatter and propidium iodide uptake characteristics of our cell preparations to cell debris, erythrocytes as well as viable and non-viable oligodendrocytes. By gating on a multiparameter basis on both defined forward light scatter and positive galactocerebroside immunolabelling, oligodendrocyte populations exceeding 98% purity were obtained which were suitable for long-term culture and further immunological and neurobiological studies.

Analysis and isolation of embryonic mammalian neurons by fluorescence- activated cell sorting

Cells were dissociated from the CNS of the embryonic mouse and rat to produce cell suspensions suitable for analysis and separation on a fluorescence-activated cell sorter (FACS). Cells from the spinal cord of the embryonic mouse were analyzed in the most detail. Cell suspensions generated three major peaks in histograms of forward-angle light scatter. Examination of material isolated from each peak and labeling of cell suspensions with the nonvital and supravital fluorescent dyes propidium iodide, ethidium bromide, and acridine orange demonstrated that the three peaks represented live cells, dead cells, and subcellular fragments. Passage through the cell sorter did not detectably damage live cells, as shown by light microscopy, FACS analysis, and in vitro culture of sorted cells. Neurons and glial cells collected by sorting survived at least 4 weeks in culture. Cell suspensions dissociated from the dorsal root ganglia, hippocampus, hypothalamus, cerebellum, and cerebral cortex of the embryonic mouse and from the spinal cord of the embryonic rat produced similar results. Analysis of samples prepared at different developmental stages showed that viable cells could be recovered from each of these regions throughout the important stages of neurogenesis and early cellular differentiation, but that few viable cells could be recovered from animals beyond late embryonic or early postnatal ages. Quantitative FACS analysis of monoclonal antibody A2B5, tetanus toxin and cholera toxin, and lectins binding to live dissociated cells from the embryonic spinal cord demonstrated that these cells had already developed binding sites for these cell-surface ligands by embryonic day 13. These results demonstrate that a fluorescence-activated cell sorter can be used for quantitative analysis of specific cellular properties, that FACS analysis and sorting can be used to identify and isolate live cells from many regions of the embryonic mammalian CNS during important developmental periods, and that sorted neurons and glial cells can be maintained for weeks in culture.

Antibodies reactive with class II antigens encoded for by the major histocompatibility complex inhibit human B cell activation.

Although class II antigens encoded by genes in the major histocompatibility complex (MHC) are important as recognition structures for immunoregulatory cell interactions, the precise functional role of these molecules in the biological responses of B lymphocytes is unknown. In the studies described here, we have examined the effects of six monoclonal antibodies reactive with human class II MHC antigens on B cell activation and proliferation. Peripheral blood IgM+ B cells purified by fluorescence-activated cell sorter (FACS) techniques were stimulated with anti-mu antibodies, protein A-bearing Staphylococcus aureus (SAC), or in T cell-dependent activation cultures. The B cell proliferative responses induced by these stimuli were inhibited 68 to 90% by low concentrations (1 to 5 micrograms/ml) of antibodies reactive with class II MHC antigens. Antibodies specific for DR and DQ antigens were both effective inhibitors of B cell proliferation. This inhibition was not due to the binding of antibody to B cell Fc-IgG receptors, because IgM and IgG anti-class II antibodies were equally potent as inhibitors. When responses of B cells fractionated on the basis of cell size by forward angle light scatter were analyzed, anti-DR and anti-DQ antibodies inhibited the proliferation of small, resting IgM+ cells induced by T-independent as well as T-dependent stimuli. Activation-dependent increases in B cell size and RNA synthesis were similarly inhibited. In contrast, the responses of large B cells (that had been preactivated in vivo) to T cell-derived B cell growth factors were not affected by anti-class II antibodies. These data suggest that class II MHC molecules do not serve merely as cellular interaction structures but also directly participate in early events of the B cell activation cascade that precede cell enlargement or increased RNA synthesis. After activation and expression of receptors for growth factors, however, B cell class II MHC antigens no longer mediate signals required for mitogenesis.

Fluorescence activated cell sorting (FACS) as a separation method for neurofibrillary tangles in Alzheimer's disease

We have developed a fluorescence cell sorting (FACS) method for the separation of neurofibrillary tangles from post-mortem brain from cases of Alzheimer's disease and Down's syndrome. Neurofibrillary tangles in brain homogenate were selectively labelled with the fluorescent dye congo red. Using FACS the tangles were separated from other tissue components on the basis of fluorescence and forward-angle light scatter to give a fraction highly enriched in tangles, which were identified by light and crossed polarisation microscopy. Tangles were not observed in a comparison fraction, collected at the same time, of weakly fluorescent particles of similar size to the tangles. Thus FACS provides a non-denaturing method for obtaining preparations of neurofibrillary tangles for purposes of further analysis.

Discrete stages of human thymocyte activation and maturation in vitro: correlation between phenotype and function.

Using light scatter, flow cytometric and functional analyses, three major stages in the in vitro activation of human thymocytes were defined. The earliest stage (day 0-2) was characterized by the induction of the interleukin 2 (IL2) and transferrin receptors, the T cell lineage specific Ta1 antigen, and increased reactivity with anti-T8 antibody. At this time, major changes in nuclear morphology but not cell size were observed. Early-stage thymocytes were immature with regard to T3 and cytotoxic capacities. The second stage (day 3-4) of in vitro culture was distinguished by loss of T6, maximal activation (both in cell size and nuclear morphology) and maximal expression of both transferrin and IL2 receptors. At this stage, nearly all thymocytes expressed T3, 30-70% of thymocytes were T4+T8+, and functionally, only small increases in cytotoxic capacities were observed. The third stage of maturation (day 5-7) represented thymocytes with reduced levels of activation as measured by forward and right angle light scatter analysis and declining IL2 and transferrin receptor expression. However, these thymocytes exhibited high levels of T3 antigen density, loss of T4, T8 coexpression and pronounced cytotoxic and detectable inducer function capabilities.