Nuclear RNA quantification in protoplast cell-cycle phases

Using acridine orange staining and flow cytometry the DNA and RNA levels (arbitrary units) of individual cells may be established. Here, this method has been applied to nuclei isolated from plant protoplasts during culture. The specificity of the technique has been validated for such plant material; ribonuclease markedly reduced nuclear staining without modifying the DNA histogram; ribonuclease inhibitor prevented the action of released cell nucleases; and protoplasts cultivated with actinomycin D did not synthesize RNA. First RNA synthesis was evident 18 h after Petunia hybrida protoplasts had been put into culture. An increase of RNA above a critical level was required for cells to be able to initiate DNA replication from G1, termed G1B. G2 nuclei had an RNA:DNA ratio similar to that of G1 nuclei.     

The relation between length of the cell cycle duration and RNA content in HeLa S3 cells

The cell cycle of HeLa S3 cells synchronized by hydroxyurea was investigated by flow cytometry. Metachromatic fluorochrome acridine orange was used to strain the DNA and RNA of the cells differentially. Periodic changes in the cellular DNA and RNA contents were observed through five cell cycles. The G1 and S phases of synchronized HeLa S3 cells that contained large amounts of RNA became shorter than those of cells that contained smaller amounts of RNA.     

Total cellular RNA content: correlation between flow cytometry and ultraviolet spectroscopy

Total RNA content in Chinese hamster ovary and HeLa-S3 cells determined by ultraviolet spectroscopy is compared with the red fluorescence distribution of acridine orange-stained cells observed by flow cytometry. A correlation coefficient of 0.93 is obtained when these methods of estimating RNA content are compared after various RNAse treatments. These data suggest that acridine orange staining effectively quantitates total cellular RNA content when analyzed by flow cytometry, although DNA is also shown to contribute a low but significant background of red fluorescence.     

The effects of 1,25-dihydroxyvitamin D3 on human T lymphocyte activation and proliferation: a cell cycle analysis

Recent studies have demonstrated that 1,25-dihydroxyvitamin D3 (calcitriol), the most biologically active metabolite of vitamin D, is a potent inhibitor of both lectin- and antigen-driven human T lymphocyte proliferation. To better characterize this effect, we performed cell cycle analysis of both untreated and calcitriol-treated peripheral blood mononuclear cells after PHA stimulation. By using the metachromatic dye acridine orange and flow cytometry, we found that calcitriol blocks the transition from the early, low RNA compartment of G1 (G1A) to the late, higher RNA compartment of G1 (G1B). Consistent with this observation was the inability of exogenous IL 1 or phorbol myristic acetate to overcome calcitriol's suppression of DNA synthesis. Indomethacin slightly reversed calcitriol's inhibition of transition from early to late G1, suggesting a minor, prostaglandin-dependent component to calcitriol's antiproliferative activity. Finally, by using the monoclonal antibodies anti-Tac and OKT9, we found that calcitriol had no effect on IL 2 receptor expression, an early G1 event, but markedly inhibited transferrin receptor expression, an IL 2-dependent, late G1 event. Thus, analysis of calcitriol's effects on the expression of these T cell activation antigens provides further evidence of the cell cycle specificity of calcitriol's action in regulating human T lymphocyte proliferation.     

Flow cytometric estimation of DNA and RNA content in intact cells stained with Hoechst 33342 and pyronin Y

The addition of RNA content estimation to flow cytometric measurement of DNA content provides valuable information concerning cells' transitions between quiescent and proliferative states. Equilibrium staining methods employing acridine orange have been used for DNA/RNA content measurement but are difficult to apply to intact cells and impractical for use in conjunction with fluorescent antibodies or ligands for demonstration of cell surface structures. I have used a combination of Hoechst 33342 (HO342) and pyronin Y (PY) to stain intact cells for DNA/RNA content estimation with a dual source flow cytometer using UV and blue-green or green excitation, measuring HO342 fluorescence at 430--470 nm and PY fluorescence at 590--650 nm. Results obtained with cultured cells and stimulated lymphocytes are in good agreement with those obtained using acridine orange for DNA/RNA staining; about half of the PY fluorescence can be removed from ethanol-fixed cells stained with HO342 and PY by RNAse digestion. The HO342/PY method can be combined with fluorescein immunofluorescence for detection of cell surface markers. HO342 can be combined with other tricyclic heteroaromatic dyes for DNA/RNA estimation; the combination of HO342 and oxazine 1 can be excited in a dual source instrument using a mercury arc lamp and a helium-neon laser. The staining procedure is simple; cells in medium are incubated with 5 microM HO342 at 37 degrees C for 45 min, 5 microM PY (or oxazine 1) is then added and cells are analyzed without washing after an additional 45 min incubation. Suitability of these dye combinations for vital cell staining and sorting remains to be determined.     

Discrimination of cycling and non-cycling lymphocytes by BUdR-suppressed acridine orange fluorescence in a flow cytometric system.

Stimulated lymphocytes which pass through the cell cycle may be distinguished from dormant G0 lymphocytes rapidly by flow cytometry. The method is based on cell incubation with 5-bromodeoxyuridine (BUdR) and their subsequent staining with acridine orange under conditions in which cellular DNA and RNA stain differentially. The DNA-specific green fluorescence of stimulated, cycling cells is suppressed while RNA-specific red fluorescence is affected only minimally. It is possible, therefore, to distinguish cycling vs non-cycling cells based on two entirely different parameters, i.e. BUdR incorporation and RNA content.     

RNA dependence in the cell cycle of V79 cells

The cell cycle of V79 Chinese hamster lung cells synchronized by hydroxyurea was investigated by flow cytometry. The metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA of V79 cells. Green and red fluorescence from individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry. Periodic changes of cellular DNA and RNA contents were observed over nine cell cycles. The duration of G1, S, and G2 + M phases of synchronized V79 cells whose RNA content was close to that of the cells in balanced growth was 3, 4.5, and 1.5 hours, respectively. The duration of G1 and S phases of cells containing RNA above a certain threshold was inversely proportional to the RNA content. The RNA content of cells containing RNA above the normal level regressed to normal after a few generations. Coefficients of variation for RNA content were significantly larger than those for DNA. An explanation for the decay of synchrony in a synchronized cell population is proposed.     

Rapid identification and detection of parasitized human red cells by automated flow cytometry

Rapid and reliable identification of various human red cells parasites is important in many chemotherapeutic and immunologic studies. Because manual microscopic counting is tedious and imprecise, we have developed a simple diagnostic procedure for the automated flow cytometric detection of in vitro infected red cells, using a nucleic acid-binding fluorescent dye, acridine orange. Human malaria (Plasmodium falciparum)-infected red cells from continuous human erythrocyte culture were incubated at room temperature in acridine orange stain for 5 min after which the samples were analyzed by flow cytometry. Since mature red cells contain no DNA, infected red cells were identified with a distinct fluorescent signal. A total of 200,000 cells per sample were counted and analyzed in less than 2 min. Rings, trophozoites, and schizonts were assessed and identified in synchronized infected red cell cultures by flow cytometry. In addition, various stages of infected red cells were isolated with a cell sorter. This rapid method permits accurate and reliable assessment of data with the exclusion of anomalous data such as damaged cells, extraneous material, and contaminating particles.     

The ratio of RNA to total nucleic acid content as a quantitative measure of unbalanced cell growth

Use of the metachromatic dye, acridine orange, to stain cells in suspension for flow cytometry allows for the simultaneous measurement of DNA and RNA content in individual cells. The relative RNA content as a function of total cellular nucleic acid content [alpha r = RNA/(RNA + DNA)] is a constant value, characteristic for particular cell lines during their exponential growth under optimal conditions. This ratio can be estimated for the G1A, G1B, S, and G2 + M cell cycle compartments. Changes in growth rate or the addition of antitumor drugs induces characteristic changes in the ratio either evenly throughout or at a particular phase of the cell cycle. Under such conditions, measurement of cellular DNA and RNA content provides a sensitive assay of any deviation from balanced cell growth. Unbalanced growth caused by suboptimal culture conditions or as a result of incubation with various antitumor agents is illustrated. Examples of unbalanced growth which are not correlated with cell viability as measured by cell clonogenicity are discussed.     

Cellular content of ribosomal RNA in relation to the progression and competence signals governing proliferation of 3T3 and SV40-3T3 cells

The method for differential fluorescence staining of cellular RNA and DNA by acridine orange (AO) was optimized for 3T3 and SV40-3T3 cells. Cellular contents of DNA and of ribosomal RNA (rRNA) were determined by dual-channel flow cytometry during cell-density-dependent proliferation and after stimulation of quiescent cells. With increasing density of 3T3 cells, cellular content of rRNA decreases by about 60%, whereas SV40-3T3 cells do not exhibit a significant dependence of rRNA content on cell density. 3T3 cells stimulated early after becoming quiescent resume reaccumulation of rRNA after a delay of only 4 h, whereas cells maintained at quiescence for several days exhibit a delay of about 12 h before a significant rise of rRNA is observed. The extent of rise of cellular rRNA content after different regimens of stimulation of quiescent 3T3 cells does not correlate well with the fraction of cells entering the cell cycle. These and other reported instances of discordance between rRNA content and stimulation into the cell cycle are resolved by showing that of the two signals governing entry into the cell cycle only the progression signal, but not the competence signal is associated with reaccumulation of cellular rRNA. The present results are consistent with the progression function being in essence the achievement of a threshold number of ribosomes per cell, which in conjunction with the competence signal is sufficient for initiation of the cell cycle.     

Survival of Aeromonas salmonicida in lake water.

The survival of Aeromonas salmonicida subsp. salmonicida in lake water was investigated by using a variety of techniques. They included acridine orange epifluorescence, respiration, cell culture, cell revival, flow cytometry, plasmid maintenance, and membrane fatty acid analysis. During a 21-day study, A. salmonicida became nonculturable in sterile lake water samples. Flow cytometry and direct microscopy indicated that cells were present. Although the nonculturable cells could not be revived, the recovery method did indicate that the presence of low numbers of culturable cells within samples could produce misleading results. Plasmid DNA, genomic DNA, and RNA were maintained in the nonculturable cells; in addition, changes in the fatty acid profiles were also detected. Although viability could not be proven, it was shown that the morphological integrity of nonculturable cells was maintained.     

Survival of Aeromonas salmonicida in lake water

The survival of Aeromonas salmonicida subsp. salmonicida in lake water was investigated by using a variety of techniques. They included acridine orange epifluorescence, respiration, cell culture, cell revival, flow cytometry, plasmid maintenance, and membrane fatty acid analysis. During a 21-day study, A. salmonicida became nonculturable in sterile lake water samples. Flow cytometry and direct microscopy indicated that cells were present. Although the nonculturable cells could not be revived, the recovery method did indicate that the presence of low numbers of culturable cells within samples could produce misleading results. Plasmid DNA, genomic DNA, and RNA were maintained in the nonculturable cells; in addition, changes in the fatty acid profiles were also detected. Although viability could not be proven, it was shown that the morphological integrity of nonculturable cells was maintained.     

Enumeration and Biomass Estimation of Bacteria in Aquifer Microcosm Studies by Flow Cytometry

Flow cytometry was used to enumerate and characterize bacteria from a sand column microcosm simulating aquifer conditions. Pure cultures of a species of Bacillus isolated from subsurface sediments or Bacillus megaterium were first evaluated to identify these organisms' characteristic histograms. Counting was then carried out with samples from the aquifer microcosms. Enumeration by flow cytometry was compared with more-traditional acridine orange direct counting. These two techniques gave statistically similar results. However, counting by flow cytometry, in this case, surveyed a sample size 700 times greater than did acridine orange direct counting (25 (mu)l versus 0.034 (mu)l) and required 1/10 the time (2 h versus 20 h). Flow cytometry was able to distinguish the same species of bacteria grown under different nutrient conditions, and it could distinguish changes in cell growth patterns, specifically single cell growth versus chained cell growth in different regions of an aquifer microcosm. A biomass estimate was calculated by calibrating the total fluorescence of a sample from a pure culture with the dry weight of a freeze-dried volume from the original pure culture. Growth conditions significantly affected histograms and biomass estimates, so the calibration was carried out with cells grown under conditions similar to those in the aquifer microcosm. Costs associated with using flow cytometry were minimal compared with the amount of time saved in counting cells and estimating biomass.     

Single-cell analysis demonstrates how nutrient deprivation creates apoptotic and quiescent cell populations in tumor cylindroids

Understanding how quiescent and apoptotic populations form in tumors is necessary because these cell types can considerably diminish therapeutic efficacy. Most cancer therapeutics are ineffective against quiescent cells because they target rapidly proliferating cells. Distinguishing apoptosis is important because apoptotic cells are committed to death and do not require treatment. Regrowth of quiescent cell can lead to tumor re-occurrence and metastasis, which are the leading causes of cancer mortality. We hypothesized that cylindroid cultures and acridine orange staining could be used to determine how nutrient diffusion creates apoptotic and quiescent regions in tumors. To test this hypothesis we developed a microscopy technique to measure cellular DNA and RNA content in single cells using thin cylindroids and acridine orange staining. Cell classification was compared to flow cytometry of cells grown in defined monolayer cultures. The presence of apoptosis was confirmed by morphological nuclear analysis. The effect of diffusion was determined by varying incubation time, cylindroid size, and exposing cylindroids to nutrient-deficient media. Four overlapping regions were identified as a function of cylindroid radius: an outer viable/quiescent region; a second quiescent/apoptotic region; a third late-stage apoptotic region; and an inner dead region. In monolayer cultures the absence of glutamine and growth factors induced apoptosis and hypoxia induced quiescence. Treating with nutrient-deficient media suggested that cells became quiescent near the periphery because of glucose and oxygen limitations, and became apoptotic and died further from the edge because of glutamine and growth factor limitations. These results show that cellular microenvironments can be identified in cylindroids using simple acridine orange staining and that single cell fluorescence can be measured in three-dimensional culture. The developed techniques will be useful for developing cancer therapies and determining how cell death and apoptosis are induced in three-dimensional tumor tissue.     

Identification of polymorphonuclear leukocytes in cytologic samples for flow cytometry.

Inflammatory cells are commonly present in cytologic specimens obtained for flow cytometry, and may interfere with the analysis of epithelial cells. We have found that detergent (Triton X-100) pretreatment in the two-step acridine orange staining procedure disrupts granulocyte cell membranes to yield bare nuclei; bladder epithelial and squamous cells on the other hand are quite resistant to the detergent treatment. Being deprived of their cytoplasmic RNA, the granulocytes lose red fluorescence. Moreover, the shearing forces in the cytometer extend the multisegmented granulocyte nuclei and align them in the direction of flow. Thus, they present as elongated objects in the measuring system, giving a large DNA fluorescence pulsewidth (nuclear size). These two phenomena make it possible to identify granulocytes in the recorded data, where they are discernible from the mononucleated leukocytes and from epithelial cells. By data selection the granulocytes can be excluded, rendering epithelial cell populations more amenable to analysis. This method may make it unnecessary to remove physically leukocytes from the specimen before flow cytometry; it may also provide a way to analyze the morphology of granulocyte nuclei and to assess methods to manipulate their membrane stability. Full protection from membrane disruption is accomplished by alcohol fixation, and partial protection by 20-30% serum.     

Size-dependent B lymphocyte subpopulations: relationship of cell volume to surface phenotype, cell cycle, proliferative response, and requirements for antibody production to TNP-Ficoll and TNP-BA

Murine splenic B lymphocytes were separated into size-dependent subpopulations by using counterflow centrifugation. Spleen cells were rigorously depleted of T lymphocytes to yield a population of cells that were greater than 90% surface immunoglobulin (Ig)-positive and that had a mean cell volume of 136.6 +/- 3.3 microns. From this population, five fractions of cells were obtained with mean cell volumes that ranged from 115.8 +/- 3.7 microns in fraction 1 to 168.0 +/- 6 microns in fraction 5. The cells in these five subpopulations were characterized by analysis on a fluorescence-activated cell sorter after staining with acridine orange to evaluate RNA and DNA content, and with fluorescein-conjugated anti-mu, anti-delta, and anti-Ia antibodies to evaluate their surface membrane phenotypes. DNA analysis revealed that virtually all of the cells in fractions 1 to 4 had 2 N DNA. Between 7 and 21% of fraction 5 cells were either in S-phase or contained 4 N DNA. In contrast, RNA content increased through the fractions, suggesting a transition from G0 to G1 in the subpopulations with increasing B cell size. As another measure of cell activation seen with increasing cell size, we observed a progressive increase in the expression of surface Ia and a decrease in the expression of surface IgD. In the absence of in vitro stimulation, the larger cells showed significantly higher levels of thymidine incorporation. When polyclonal B cell activators such as LPS or anti-Ig antibody were added, peak proliferative responses were similar in all of the fractions, but the time necessary to achieve a maximal response was shorter for the larger-sized cell subpopulations than it was for the smaller-sized cell subpopulations. Unprimed, size-dependent B lymphocyte subpopulations exhibited spontaneous or "background" antibody formation that occurred primarily in the subpopulations containing the largest cells. T cell factors present in EL4 supernatant enhanced the efficiency of in vitro differentiation of these same subpopulations. When cultured in the absence of T cell help, the thymus-independent type 1 (TI-1) antigen TNP-Brucella abortus (TNP-BA) or the thymus-independent type 2 (TI-2) antigen TNP-Ficoll induced the largest anti-TNP plaque-forming cell (PFC) responses in the fractions containing intermediate-sized cells, suggesting that in vitro, antigen-specific responses came primarily from B cells that have been influenced in vivo to leave their small resting state. The subpopulations containing the smallest size B cells required the presence of both a TI antigen and EL4 supernatant for efficient differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Control of cellular proliferation in HeLa-S3 suspension cultures. Characterization of cultures utilizing acridine orange staining procedures

Growth control is investigated in detail in fed and unfed HeLa-S3 suspension cultures. Two-step acridine orange staining and flow cytometric analysis indicated declines in cellular red fluorescence (proportional to RNA content) of 40-50% between exponential and plateau phase in both culture types. Cellular green fluorescence (DNA content) assessed simultaneously indicates an increment of cells with Gi-DNA content in plateau phase in the unfed cultures, while fed cultures show a brief increment in G1-phase cells in the transition phase followed by a recovery in plateau phase to a value similar to that of exponential cultures. Temporal declines in the 3H-thymidine pulse-labeling index are observed in both culture systems. These data along with the flow cytometry data indicate a distinct G1-arrest in the unfed plateau cultures and suggest a random arrest of cells about the cell cycle in fed plateau cultures. Acidic acridine orange staining and flow cytometric analysis furthermore indicate the occurrence of a quiescent population comprising approximately 345 of the total cells and consisting of both dead and viable cells in plateau phase unfed cultures. In contrast, fed plateau cultures show approximately 14% quiescent, mostly dead cells. Also, both culture systems show temporal declines in the clonogenic index and a longer cell-cycle transit time in plateau phase relative to exponential phase. These findings confirm earlier work which indicates that the environment has a profound influence on the mode of growth control for mammalian cells in vitro.     

Quantitation of lymphocyte response to antigen by flow cytofluorometry.

The response of human peripheral blood lymphocytes to antigenic stimulation has been studied in vitro using flow cytofluorometry and an acridine orange (AO) staining technique for cellular deoxyribonucleic acid and ribonucleic acid (RNA). Antigen-stimulated "pyroninophillic" immunoblasts, identified by an increase in cellular content of RNA (red fluorescence with AO), were quantitated in triplicate cultures incubated up to 7 days with and without bacterial antigen. These results were similar to 14C-thymidine incorporation into identical cultures incubated in parallel. Cytofluorometric analysis showed a peak in percentage of immunoblasts after 6 days in culture, while maximum thymidine incorporation was seen on day 7. Cells from patients with depressed immune response secondary to cancer showed lower than normal antigen response by cytofluorometry. Kinetic studies revealed both a lower percentage of immunoblasts when compared to normal and a lower average per cell RNA content of the stimulated cells. AO cytofluorometry is suggested as a convenient method of simultaneously assessing lymphocyte proliferative and nonproliferative response to antigen.     

Chromatin condensation during spermiogenesis in the golden hamster (Mesocricetus aureus): a flow cytometric study

DNA-staining of hamster testis cell suspensions followed by flow cytometry demonstrated appearance of the first haploid cells at 23 days post partum (dpp) and of condensed chromatin (in elongated spermatids and spermatozoa) at 33-34 dpp. Mature spermatozoa were first observed in the caput epididymis at 36-37 dpp, thus completing the first spermatogenic wave. Testicular cell suspensions from animals from 23 to 38 dpp were stained with acridine orange, and flow cytometer gating was adjusted to include only the haploid cells. Acridine orange intercalated into double-stranded DNA to produce green fluorescence. The decrease in green fluorescence intensity from 23 until 37 dpp was caused by changes in the binding of DNA to basic proteins in such a fashion as to impede the access of the dye to the DNA double helix. When the green fluorescence values (of the most advanced spermatids) were plotted against the age of the hamsters (in dpp) or the corresponding steps of spermiogenesis, the decrease in fluorescence could be seen to occur in three phases. The inflection point between the first and second phases was observed at about spermiogenesis step 7, consistent with the hypothesis that this represents removal of histone from the chromatin. The second phase presumably represents the period in which transition proteins are bound to the DNA. At approximately steps 15 or 16 a further inflection point was seen where protamines replaced the transition proteins. The red fluorescence produced when acridine orange bound to RNA in spermatids, increased early in spermiogenesis and decreased dramatically at 34 dpp, consistent with the fact that elongating spermatids discard the bulk of their cytoplasm during the maturation process.