Reversal of sodium-azide mutagenicity by liver preparations and by gastric juice.

Sodium azide was found to be mutagenic for Salmonella typhimurium by inducing base-pair substitutions that were not enhanced by pKM101 plasmid (R factor). However, the mutagenicity of sodium azide was decreased by enzyme proteins contained in rat-liver post-mitochondrial fractions, depending on the NADPH-generating system. Pre-incubation with human gastric juice also decreased azide mutagenicity. These metabolic effects might explain the conflicting nature of the mutagenicity and carcinogenicity tests reported in the literature. Laboratory reagents containing 0.1% sodium azide as a preservative showed the expected patterns of mutagenicity and of metabolic deactivation, and no aspecific interaction could be detected between azide and the various components, including proteins, of the reagents tested.     

On the reaction of sulfenyl iodide derivatives with azide

Based on the suggested mechanism of the Raschig catalytic iodine-azide reaction the use of azide for the azotometric estimation of sulfenyl iodide groups is proposed. In the Raschig reaction reduction of iodine to iodide and oxidation of azide to elementary nitrogen is specifically catalyzed by bivalent sulfur compounds; the reaction is usually formulated to proceed via hypothetical sulfenyl iodide derivatives. This has been explored with the use of available, relatively stable sulfenyl iodide derivatives. The -SI group oxidizes azide to nitrogen stoichiometrically: 1 mole of a sulfenyl iodide consumes 2 moles of sodium azide and yields 3 moles of elementary nitrogen. The specificity and limitations of the method are discussed.     

Quantification and Characterization of Phagocytosis in the Soil Amoeba Acanthamoeba castellanii by Flow Cytometry

Phagocytosis in the common grazing soil amoeba Acanthamoeba castellanii was characterized by flow cytometry. Uptake of fluorescently labelled latex microbeads by cells was quantified by appropriate setting of thresholds on light scatter channels and, subsequently, on fluorescence histograms. Confocal laser scanning microscopy was used to verify the effectiveness of sodium azide as a control for distinguishing between cell surface binding and internalization of beads. It was found that binding of beads at the cell surface was complete within 5 min and 80% of cells had beads associated with them after 10 min. However, the total number of phagocytosed beads continued to rise up to 2 h. The prolonged increase in numbers of beads phagocytosed was due to cell populations containing increasing numbers of beads peaking at increasing time intervals from the onset of phagocytosis. Fine adjustment of thresholds on light scatter channels was used to fractionate cells according to cell volume (cell cycle stage). Phagocytotic activity was approximately threefold higher in the largest (oldest) than in the smallest (newly divided) cells of A. castellanii and showed some evidence of periodicity. At no stage in the cell cycle did phagocytosis cease. Binding and phagocytosis of beads were also markedly influenced by culture age and rate of rotary agitation of cell suspensions. Saturation of phagocytosis (per cell) at increasing bead or decreasing cell concentrations occurred at bead/cell ratios exceeding 10:1. This was probably a result of a limitation of the vacuolar uptake system of A. castellanii, as no saturation of bead binding was evident. The advantages of flow cytometry for characterization of phagocytosis at the single-cell level in heterogeneous protozoal populations and the significance of the present results are discussed.     

Computer simulations of the effect of non-inactivating sodium channels on the electric behavior of excitable cells

Non-inactivating sodium channels have been discovered in various cell types. Additionally, normal voltage-gated sodium channels can be induced to lose their ability to inactivate by treatment with proteolytic enzymes, with certain chemical reagents, or with toxins. The presence of non-inactivating sodium channels in the outer membrane of a cell is expected to profoundly modify the electrical properties of the cell, because the electrical depolarization of the cell and the opening of these channels reciprocally reinforce each other without intrinsic control. The normal resting state may thus be destabilized and a new resting state at depolarized resting potentials may become possible. In this study, computer simulations were carried out to systematically explore the patterns of behavior of excitable cells which have non-inactivating sodium channels in their plasma membrane. The cells were assumed to be space clamped and the relevant Hodgkin and Huxley equations were assumed to describe the electrical behavior of the cells, except that some or all of the sodium channels could not inactivate. The sodium currents were thus represented by the sum of two terms: FI.gNa.m3.h.(V-ENa) + (1-FI).gNa.m3(V-ENa), where FI(0 less than or equal to FI less than or equal to 1) is the fraction of sodium channels which inactivate normally, and the other symbols have their usual significance. The behavior of non-inactivating sodium channels created by pronase treatment or reaction with chemical reagents was found to conform with that predicted by the second term in this expression. The simulations thus quantitatively apply to excitable cells thus treated, but may serve additionally to qualitatively illustrate patterns of electrical activity induced by non-inactivating sodium channels also in other cases. A variety of possible types of electrical behavior was obtained: Normal behavior, including capability of firing action potentials, requires values of FI which are not far from unity, the permissible range depending on the fully activated potassium ion conductance, gK. Bistability, at which the cell may exist in one of two stable states of different resting potential, occurs when the value of FI is lowered. Transitions from the polarized to the depolarized resting states, and vice versa, may be brought about by depolarizing and hyperpolarizing triggers, respectively. Such behavior is like that of memory storage devices. Monostability at depolarized potentials is favored by low FI values and can occur if gK is less than the Hodgkin and Huxley value.(ABSTRACT TRUNCATED AT 400 WORDS)     

Method to overcome photoreaction,a serious drawback to the use of dichlorofluorescin in evaluation of reactive oxygen species

Non-fluorescent dichlorofluorescin (DCFH) was converted to fluorescent products by photo-irradiation during observations with spectrofluorometer and fluorescence microscopy. Photo-irradiation of DCFH at 250, 300, 330, 400, 500, or 600 nm generated fluorescent dichlorofluorescein (DCF), an oxidation product of DCFH, and an unrecognized fluorescent product. The ratio of the unknown product to DCF varied from 0.15 to 8.21 depending on wavelength. Although reactive oxygen species scavengers, such as catalase, superoxide dismutase, and sodium azide, did not suppress the increase in non-specified fluorescence, reagents such as ascorbic acid, mercaptopropionyl glycine, and methoxycinnamic acid, in a cell-free system, almost completely suppressed it with little effect on the fluorescence of DCF. Meanwhile, ascorbic acid also suppressed non-specified fluorescence in cells, but not completely. At low concentrations of DCFH, the speed of increasing fluorescence was considerably retarded, to such a degree that the fluorescence increase in cells during fluorescence microscopic observation was negligible. The addition, at the time of evaluation, of the above reagents to cell-free systems and, in cell systems, reducing the concentration of DCFH, effectively suppressed the photoreaction of DCFH.     

Protein enrichment by capture-release based on strain-promoted cycloaddition of azide with bicyclononyne (BCN)

An enrichment strategy was devised for azide derivatized macromolecules, based on strain-promoted alkyne-azide cycloaddition (SPAAC) and a cleavable linker. A ring-strained alkyne, bicyclo[6.1.0]non-4-yne (BCN), was covalently attached to agarose beads via a hydrazine-sensitive linker. Benchmark studies of the resulting 'azido-trap' beads were performed with a fluorogenic coumarin derivative, leading to efficient capture of the azidocoumarin with concomitant fluorescence staining of the beads via SPAAC. The versatility of the beads for specific protein enrichment was shown by an effective and highly specific capture-release strategy for enrichment of azido-containing Candida antarctica lipase B (CalB) from a mixture of proteins. This approach is suited for selective enrichment of (glyco)proteins after metabolic incorporation of azides for subsequent (glyco)proteomics studies.     

A versatile transition metal salt reaction for a wide range of common biochemical reagents: an instantaneous and quantifiable color test

A rapid and sensitive spot test amenable to visual or spectrophotometric quantitation has been developed for a wide variety of biochemical reagents by utilizing the transition metal salt cupric chloride and its large number of related colored compounds. This assay is potentially a widely applicable multipurpose test for rapidly detecting the presence of unknown substances. Combination of the test sample with the working reagent results in the immediate formation of a distinctive colored product that may be precipitable. Some compounds require the further addition of sodium hydroxide in order to generate the distinctively colored product. Distinctive reactions occur with the following reagents, and their limit of visual detection is indicated in parentheses: ammonium bicarbonate (12.5 mM), ammonium acetate (25 mM), ammonium hydroxide (0.1%), ammonium sulfate (2%), ammonium persulfate (0.02 mM), L-(+)-cysteine (0.07 mM), dithiothreitol (DTT) (1.25 mM), EDTA (0.6 mM), ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (5 mM), D-glucose (6 mM), glycerol (0.3%), imidazol (12.5 mM), DL-methionine (100 mM), mercaptoethanol (0.05%), sodium azide (19 mM, 0.1%), sodium dithionite (0.25%), sodium metabisulfite (25 mM), sodium nitrite (6.2 mM), sodium periodate (3.1 mM), sodium sulfite (12.5 mM), sodium thiosulfite (12.5 mM), sucrose (6 mM), and N,N,N',N'-tetramethylethylenediamine (0.05%). A distinctive exothermic reaction occurs with hydrogen peroxide, but without color change. Compounds reacting insignificantly include 50 mM Tris buffer, urea, N,N'-methylene bisacrylamide, sodium dodecyl sulfate, isopropyl alcohol, sodium fluoride, trichloroacetic acid, phenol, mannose, K2HPO4, guanidine HCl, chloramine-T, magnesium chloride, and boric acid, where the solids were tested at approximately 10 mg/ml. Spectrophotometric standard curves were developed for DTT and sodium azide utilizing the clear supernatants resulting from these reactions. Combinations of at least four reagents could be discriminated, as demonstrated with mixtures of glucose, sodium azide, EDTA, and DTT. In addition ammonium sulfate could be detected to a limit of 4% in the presence of protein, DTT, and EDTA in a 50 mM Tris buffer. Spot tests were developed which utilized reagent-impregnated filter paper and gave distinctive colored products on addition of 5 microliter of test sample.     

Phagocytic Activity of FRTL-5 Rat Thyroid Follicular Cells as Measured by Ingestion of Fluorescent Latex Beads

A rat thyroid cell line (FRTL-5) was used to study the phagocytic activity of thyroid follicular cells using fluorescent latex beads and flow cytometric analysis. Morphologic studies demonstrated that latex beads were engulfed and located within cytoplasmic vacuoles of thyrocytes. Flow cytometric evaluation of cell suspensions revealed high levels of fluorescence in cells engulfing latex beads. Using thyrotropin (TSH) as a stimulator of thyroid function and human interleukin-1β as an inhibitor, protocols were established for measuring the effects of these substances on either basal or TSH-induced phagocytosis. Cells exposed to latex beads over time in basal (0H) or TSH-containing medium had an increase in time-dependent phagocytic activity which was maximal after 24 or 8 h, respectively. Treatment of FRTL-5 cells with either a stimulator or an inhibitor revealed maximal change in phagocytic activity after 72 h as measured by the percentage of phagocytic cells as well as the mean fluorescence intensity. Phagocytic activity and iodide trapping by FRTL-5 cells were qualitatively similar in both sensitivity and magnitude of change in the assays used in this study. Phagocytosis of fluorescent latex beads represents a sensitive nonradioactive assay of thyrocyte function whose regulation is similar to iodide trapping.     

Increased immunofluorescence sensitivity using 532 nm laser excitation.

OBJECTIVE: We evaluated the use of a high power, diode pulsed solid-state laser emitting 532 nm light for immunofluorescence applications. We compared the sensitivity and utility of this laser with the standard 488 nm excitation. METHODS: A flow cytometer was equipped with both a 488 nm and a 532 nm laser; fluorescence emissions from each laser were collected using the same filters and the same detector system. Cells or compensation beads (e.g. latex beads coated with anti-kappa antibodies) were stained with monoclonal antibodies conjugated to phycoerythrin (PE) as well as the PE tandem dyes TRPE, Cy5PE, Cy5.5PE, and Cy7PE. The sensitivity of detection of these reagents as well as those in heavily compensated channels was quantified by measuring the spreading error for a primary detector into a secondary detector. RESULTS: Measurement of the fluorescence emission of PE and PE-tandem dyes was considerably more sensitive when using 532 nm excitation (150 mW) as compared with 488 nm excitation (20 mW). In addition, as the absolute number of photoelectrons collected was greater, there was less measurement-error-induced spread into the compensated channels. As an example, when comparing the spreading error of PE labeled cells into the TRPE detector, the green laser was found to be 15-fold more sensitive as compared with the blue laser. In addition, the blue laser produced more autofluoresent signal from cells as compared with the green laser. Together, these advantages of the 532 nm excitation line provides for a significantly improved detection of immunofluorescence staining.     

Retardation of immunofluorescence fading during microscopy

Polyvinyl alcohol (PVA) mounting medium containing paraphenylenediamine (PPD), n-propyl gallate (NPG), or 1,4-diazobicyclo(2,2,2)-octane (DABCO) was compared with PVA alone or buffered glycerol with regard to capacity for preservation of immunofluorescence preparations. The results were based on staining of an artificial substrate with homogeneous antigen distribution followed by microphotometric determination of the initial light emission from bound fluorescein isothiocyanate (FITC)-labeled antibody and the subsequent fluorescence fading during 3-min exposure to blue excitation light. At a concentration of 0.2-2.0 g/liter and 6 g/liter, respectively, PPD and NPG were shown to effectively retard fluorescence fading without notably decreasing the initial emission intensity; two requisites were that the modified PVA used must be rather fresh and that the mounted preparations be examined within a few days. Although addition of DABCO (6 g/liter) afforded a mounting medium that tolerated storage before use better, but both PPD and NPG were more advantageous in practice. The retarding effect of PPD on fading of FITC emission was confirmed by performance testing on human tissue sections. Remounting in PVA alone is recommended for prolonged storage of sections that have been mounted in PVA modified with one of the above-mentioned compounds.     

Fluorescence properties and conformation of human milk bile salt-activated lipase

The fluorescence properties of human milk bile salt-activated lipase (BAL) in aqueous solution at various pH and in the presence of denaturing reagents and bile salts have been studied by measuring the accessibility of tryptophan side chains to the iodide ion. The fluoresence quenching studies of BAL demonstrated that the BAL conformation was pH sensitive. At pH 7.5, in the presence of denaturing reagents, all of the BAL tryptophan became accessible to iodide, suggesting the presence of random conformation in this medium. The decrease in tryptophan accessibility to iodide with various bile salt activators was found to correlate with the corresponding activity of BAL with long chain triacylglycerol substrate.     

Cr(VI) reduction by Pseudomonas aeruginosa immobilized in a polyvinyl alcohol/sodium alginate matrix containing multi-walled carbon nanotubes

Pseudomonas aeruginosa (P. aeruginosa) was immobilized with polyvinyl alcohol (PVA), sodium alginate and multiwalled carbon nanotubes (MCNTs). After immobilization, the beads were subjected to freeze-thawing to enhance mechanical strength. When exposed to 80 mg/L Cr(VI), the immobilized bacteria were able to reduce 50% of them in 84 h, however the free cells were deactivated at this concentration. The beads were used to reduce 50 mg/L Cr(VI) for nine times, with the reduction efficiency above 90% in the first five times and 65% in the end.     

Flow cytometric evaluation of proliferative activity and ploidy in myelodysplastic syndromes and acute leukemias

Propidium iodide (PI) DNA distribution of bone marrow (BM) cells was studied by flow cytometry (FCM) in 36 patients without hematologic or malignant disease (normal BM) and in 172 patients with anemias (36 pts), myelodysplastic syndromes (MDS) (33 pts) and acute leukemia (AL) at diagnosis (60 pts), remission (24 pts) and relapse (19 pts). White blood cells from normal male subjects were used as an external diploid reference standard (median CV = 3.8). Patients with normal BM, anemias, MDS and acute leukemia at diagnosis had tritiated thymidine labeling index (LI) and most with MDS and AL had also evaluable cytogenetics performed on the same BM sample used for FCM. In normal BM, median aliquot of cells with PI-DNA content intermediate between the diploid and the tetraploid value (2n-4n cells %) was 15.7. The ratio between the fluorescence intensity of the G0/1 peak of normal BM cells and the fluorescence intensity of the G0/1 peak of the reference standard (FI ratio) ranged from 93 to 1.05 (mean +/- 2SD). The 2n-4n cell % was higher than normal in anemias (p less than .001), lower in leukemias (p less than .001) and widely scattered in MDS. A linear correlation was found between 2n-4n cell % and LI, with 2n-4n cell % value higher than LI. The FI ratio was lower than normal in anemias (p less than .05), higher in AL with normal cytogenetics (p less than .02) and broadly scattered in MDS with normal cytogenetics. From our experience, PI-DNA-FCM is a simple and adequate method to evaluate proliferative activity in hematologic diseases. Nevertheless, caution must be taken in attributing small changes in FI ratio to aneuploidy, since they are found in anemias and in MDS and AL with normal cytogenetics, possibly due to differences in PI uptake by different cell types.     

Modelling of Mg2+, ATP-dependent mitochondrial Ca ions transport in smooth muscle cells using protonophore CCCP-sensitive fluorescent tetracycline

Mg2+, ATP-dependent Ca2+ accumulation in the rat myometrial mitochondria was investigated in complex experiment using Ca2+ isotope (45Ca2+) and Ca(2+)-sensitive label tetracycline. Monotonous increase of the fluorescence signal, insensitive to thapsigargin (100 nM) was observed with following establishing the stationary state of incubation at 2 min. which correlates with results obtained using isotope technique. Experiments with isotope label signify, that protonophore CCCP, ruthenium red and sodium azide, in concentration 1 microM, 10 microM and 10 mM respectively, totally inhibits the accumulation of the Ca ions in mitochondria. At the same time, in conditions of Mg2+, ATP-dependent Ca2+ accumulation modeling in these cellular structures, CCCP and sodium azide, used in the same concentration, diminished tetracycline fluorescence signal increase. In the same conditions, the introduction of the CCCP (1 mM) into the incubation medium at 75 sec. after initiation of the transport process induced reversible quenching of the tetracycline fluorescence signal to the level, observed in case of initial CCCP presence in the medium. According to data obtained in the experiment, using Ca2+ isotope, Ca(2+)-ionophore A-23187 induces both the reversible release of previously accumulated Ca ions, and cause reversible quenching of the tetracycline fluorescence signal to the level, observed in case of initial CCCP (1 mM) and sodium azide (10 mM) presence in the incubation medium. Conclusion was drawn that the thapsigargin-insensitive and CCCP, sodium azide and A-23187-sensitive tetracycline fluorescence increasing in case of modeling of Mg2+, ATP-dependent Ca2+ accumulation in myometrial mitochondria reflect the Ca2+ uniporter functioning in those subcellular structures.     

New fluorogenic, photoactivable, heterobifunctional crosslinking thiol reagents

Properties of newly synthesized crosslinking reagents (ACM) and their applications to proteins are studied (ACM is the abbreviation for a series of photoactivable and heterobifunctional crosslinking thiol reagents, each of which has two reactive groups, maleimide and azide). These reagents bind specifically to the sulfhydryl residues of proteins in the first reaction step. Upon photoactivation, the azide group of the coumarin ring reacts with side or main chains of the proteins, and thus intra- or intermolecular crosslinking can be elicited. In addition, the coumarin moiety of the reagents becomes highly fluorescent after photolysis. Therefore, the crosslinking products can be detected by fluorometry with high sensitivity in the pattern of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reaction of ACM with rabbit muscle aldolase led to extensive crosslinking between subunits of the enzyme and maximally 25% of the total subunits were found to be crosslinked to the dimer.     

A reduction-triggered fluorescence probe for sensing nucleic acids

We have developed a reduction-triggered fluorescence probe with a new fluorogenic compound derivatized from Rhodamine for sensing oligonucleotides. The chemistry to activate the compound involves the reaction between the azide group of rhodamine derivatives and the reducing reagents, with the fluorescence signal appearing after reduction of the azide group. The signal/background ratio of this fluorogenic compound reached 2100-fold enhancement in fluorescence intensity. Dithio-1,4-threitol or triphenylphosphine as reducing reagents were successfully utilized for this chemistry to be introduced into the DNA probe. The genetic detection requires that two strands of DNA bind onto target oligonucleotides, one probe carrying a reducible fluorogenic compound while the other carries the reducing reagents. The reaction proceeds automatically without any enzymes or reagents under biological conditions to produce a fluorescence signal within 10-20 min in the presence of target DNA or RNA. In addition, the probe was very stable under biological conditions, even such extreme conditions as pH 5 solution, pH 10 solution, or high temperature (90 degrees C) with no undesirable background signal. The probes were successfully applied to the detection of oligonucleotides at the single nucleotide level in solution and endogenous RNA in bacterial cells.     

Spindle microtubule dynamics: modulation by metabolic inhibitors

Recent experiments have shown that spindle microtubules are exceedingly dynamic. Measurements of fluorescence recovery after photobleaching (FRAP), in cells previously microinjected with fluorescent tubulin, provide quantitative information concerning the rate of turnover, or exchange, of tubulin subunits with the population of microtubules in living cells at steady state. In an effort to elucidate the pathways and factors that regulate tubulin exchange with microtubules in living cells, we have investigated the energy requirements for tubulin turnover as measured by FRAP. Spindle morphology was not detectably altered in cells incubated with 5 mM sodium azide and 1 mM 2-deoxyglucose (Az/DOG) for 5 minutes, as assayed by polarized light microscopy and antitubulin immunofluorescence. In FRAP experiments on these ATP-depleted cells, the average rate of recovery and the average percent of bleached fluorescence recovered were reduced to 37% and 30% of controls, respectively. When the inhibitors were removed, cells continued through mitosis, and rapid FRAP was restored. In the presence of azide and glucose, the rate of recovery and percent of fluorescence recovered were only slightly reduced, demonstrating that energy production via glycolysis can support microtubule turnover. Longer incubations with Az/DOG altered the microtubule organization in mitotic cells: astral microtubules lengthened and spindle fibers shortened. Furthermore, both astral and spindle microtubules became resistant to nocodazole-induced disassembly under these conditions. Together these observations indicate that microtubule dynamics require ATP and suggest a relationship between microtubule organization and turnover.     

Propidium iodide as an indicator of Giardia cyst viability.

The use of propidium iodide, whose uptake indicates cell death or damage, was investigated to assess the viability of heat-inactivated and chemically inactivated Giardia muris cysts. This was done by comparing propidium iodide staining with excystation. We first determined that propidium iodide could be used with an immunofluorescence detection procedure by showing that the percentages of Giardia lamblia cysts stained with this dye before and after subjecting them to a fluorescence detection method were similar. G. muris cysts were then exposed to heat (56 degrees C), 0.5 to 4 mg of chlorine per liter (pH 7.0, 5 degrees C), 0.1 to 10 mg of a quaternary ammonium compound per liter, or 2 mg of preformed and forming monochloramine per liter (pH 7.2, 18 to 20 degrees C). A good positive correlation between percent propidium iodide-stained cysts and lack of excystation was demonstrated for G. muris cysts exposed either to heat or to the quaternary ammonium compound. However, no significant correlation between absence of excystation and propidium iodide staining was found for cysts exposed to chlorine or monochloramines. These results demonstrate that the propidium iodide staining procedure is not satisfactory for determining the viability of G. muris cysts exposed to these two commonly used drinking water disinfectants.     

Fibroblast spreading and phagocytosis: similar cell responses to different-sized substrata

Experiments were carried out to test the hypothesis that cell spreading and phagocytosis are similar cell responses to different-sized substrata. The following morphological and biochemical studies provided evidence for this supposition. Cells phagocytosed 1.09-micron and 5.7-micron latex beads, but were unable to completely ingest 15.8-micron or 25.7-micron beads. With the larger beads, the cells spread around the bead surfaces with an appearance typical of cells spread on culture dishes. Biochemical studies with cytochalasin D, azide, and iodoacetate, as well as temperature-dependence studies, demonstrated similar responses of cell spreading and phagocytosis to these treatments. Similar cell surface receptors were involved in cell spreading and phagocytosis based upon experiments using antibodies to baby hamster kidney (BHK) cell wheat germ agglutinin receptors. And finally, BHK cell variants with defective plasma fibronectin (pFN) receptors were unable to spread on pFN-coated dishes or ingest pFN-coated beads. Evidence also is presented concerning the "contact" process in cell adhesion. It was found that azide and low temperature inhibited cell attachment per se but did not block fibronectin-receptor interactions based upon cell binding of pFN-coated beads. A possible explanation for the contact process is presented based upon the resistance of cells and beads to shear forces.     

The relationship between thrombomodulin expression and cell cycle stages in cultured rabbit endothelial cells

Cultured rabbit endothelial cells have significant but variable amounts of thrombomodulin (TM), both on their surface as well as inside the cell. To determine if variations in TM antigen is cell cycle related, cells with very high levels of TM antigen were identified and staged according to the intracellular distribution and relative amounts of the antigen, using immunofluorescence techniques. After staging, the nuclear DNA content of each of these cells was determined by measuring the propidium iodide (PI) fluorescence intensity cytophotometrically. Stages 1, 2, and 3, which exhibited TM immunofluorescence in the golgi area, clustered to the G1 phase of the cell cycle. Cells without discernible golgi fluorescence (stages 4 and 5) but with variable amounts of cytoplasmic and surface fluorescence appeared to have little or no relationship to the cell cycle.