Grazing Characteristics and Growth Efficiencies at Two Different Temperatures for Three Nanoflagellates Fed with Vibrio Bacteria at Three Different Concentrations

Small inocula of one of the flagellates Paraphysomonas imperforata, Pteridomonas danica, and Cafeteria roenbergensis were added to suspensions of the bacterium Vibrio natriegens at each of three concentrations between 107 and 108 cells ml-1 and incubated at each of the temperatures 10 degrees C and 25 degrees C. Samples were taken at intervals for counting the flagellates and bacteria to determine the timing of the maximum of flagellate numbers and the concentrations at that time. Measurements of the protein concentration of the suspensions during incubation were used to determine the gross growth efficiency (GGE) or yield of flagellate grazing in each experiment. The most effective grazer was Pteridomonas, followed by Paraphysomonas, with Cafeteria being least effective, as judged by the threshold bacterial concentrations at which flagellate multiplication ceased, which were about 2 x 105, 2 x 106, and 2 x 107, respectively, and by the finding that Pteridomonas consumed 99%, Paraphysomonas about 95%, and Cafeteria only 60-70% of the available bacteria in the experiments. Peak concentrations of flagellates were reached later at the lower temperature, but the numbers of flagellates produced and of bacteria eaten were of a similar order at the two temperatures and the GGE was only slightly higher at the lower temperature. The time taken to reach peak flagellate numbers changed little with a threefold increase in bacterial concentrations, but the GGE increased and the numbers of bacteria eaten to produce one flagellate decreased when the bacterial concentration was increased. The three flagellates show clear evidence of niche specialization in differences in thresholds of bacterial prey concentration.     

Comparison of growth efficiencies of protozoa growing on bacteria deposited on surfaces and in suspension

Bacteria were deposited in tubes as compact pellets by centrifuging suspensions of cultured Vibrio at stationary phase. Numbers and protein biomass of flagellates added to these tubes and of the Vibrio, were followed and compared with the growth of the same and other protists on identical, uncentrifuged Vibrio. The flagellates Bodo saliens and Caecitellus parvulus, which could not be seen to multiply in tubes of suspended bacteria, grazed deposited bacteria actively as did the more versatile flagellate Cafeteria roenbergensis. The growth of these flagellates and their consumption of deposited bacteria were very similar to those of the flagellate Pteridomonas danica or the ciliate Uronema marinum fed with suspended bacteria, although deposit-feeders grew more slowly. Gross growth efficiencies (30-60%) of deposit-feeding flagellates were similar to those of the suspension-feeding protists. Caecitellus consumed 55 Vibrio to produce one flagellate, while 4,500 Vibrio were consumed to produce one Uronema. Surface-feeding flagellates are shown to be efficient bacterivores, capable of restricting the numbers of bacteria deposited on surfaces just as other protozoa control numbers of suspended bacteria.     

Bacterivory by starved marine heterotrophic nanoflagellates of two species which feed differently, estimated by uptake of dual radioactive-labelled bacteria

Abstract: The rates of ingestion of bacteria and of accumulation of bacterial biomass by hungry Pteridomonas danica and Paraphysomonas imperforata were measured using dual radioactive-labelled bacteria in experiments lasting 4–8 h. Pteridomonas continuously consumed 4–5 bacteria h⁻¹ throughout experiments lasting 8 h, irrespective of bacterial concentration above a threshold of about 5 × 10⁵ bacteria ml⁻¹, and continued to catch bacteria even below this density. The clearance rate of about 1 nl cell⁻¹ h⁻¹ at higher bacterial concentrations increased three or four times as bacterial numbers fell. Paraphysomonas cells, with only half the biomass of Pteridomonas , ingested up to 10 bacteria h⁻¹ at high bacterial concentrations, and gradually reduced the feeding rate, effectively ceasing to feed at 10⁶ bacteria ml⁻¹; their initial clearance rate of 1–2.5 nl cell⁻¹ h⁻¹ subsequently fell as low as 0.1 nl cell⁻¹ h⁻¹. Estimation of feeding rate by extrapolation from short-term experiments on such flagellates requires extreme caution. These flagellates, starved to levels typical of the natural environment, accumulated ingested bacterial biomass at an efficiency of between 16 and 21%, indicating that in nature they would recycle 80% or more of the nutrients contained in their food.     

Flow Cytometric Analysis of Characteristics of Hybridization of Species-Specific Fluorescent Oligonucleotide Probes to rRNA of Marine Nanoflagellates

Identification problems restrict quantitative ecological research on specific nanoflagellates. Identification by specific oligonucleotide probes permits use of flow cytometry for enumeration and measurement of size of nanoflagellates in statistically meaningful samples. Flow cytometry also permits measurement of intensity of probe binding by cells. Five fluorescent probes targeted to different regions of the small subunit rRNA of the common marine flagellate Paraphysomonas vestita all hybridized with cells of this flagellate. Cells fixed with trichloroacetic acid gave detectable signals at a probe concentration of 15 aM and specific fluorescence increased almost linearly to 1.5 fM, but at higher concentrations nonspecific binding increased sharply. Three flagellates, P. vestita, Paraphysomonas imperforata, and Pteridomonas danica, all bound a general eukaryotic probe approximately in proportion to their cell size, but the specific P. vestita probe gave 14 times more fluorescence with P. vestita than with either of the other flagellates. Cell fluorescence increased during the early growth of a batch culture and decreased toward the stationary phase; cell size changed in a comparable manner. Cell fluorescence intensity may allow inferences about growth rate, but whether fluorescence (assumed to reflect ribosome number) merely correlates with cell biomass or changes in a more complex manner remains unresolved.     

Effects of pulse labelling with tritiated thymidine on the circadian rhythmicity in epidermal cell-cycle distribution in mice

The influence of pulse labelling with 50 microCi tritiated thymidine ( [3H]TdR) (2 microCi/g) on epidermal cell-cycle distribution in mice was investigated. Animals were injected intraperitoneally with the radioactive tracer or with saline at 08.00 hours, and groups of animals were sacrificed at intervals during the following 32 hr. Epidermal basal cells were isolated from the back skin of the animals and prepared for DNA flow cytometry, and the proportions of cells in the S and G2 phases of the cell cycle were estimated from the obtained DNA frequency distributions. The proportions of mitoses among basal cells were determined in histological sections from the same animals, as were the numbers of [3H]TdR-labelled cells per microscopic field by means of autoradiography. The results showed that the [3H]TdR activity did not affect the pattern of circadian rhythms in the proportions of cells in S, G2 and M phase during the first 32 hr after the injection. The number of labelled cells per vision field was approximately doubled between 8 and 12 hr after tracer injection, indicating an unperturbed cell-cycle progression of the labelled cohort. In agreement with previous reports, an increase in the mitotic index was seen during the first 2 hr. These data are in agreement with the assumption that 50 microCi [3H]TdR given as a pulse does not perturb cell-cycle progression in mouse epidermis in a way that invalidates percentage labelled mitosis (PLM) and double-labelling experiments.     

Complex flagellar motions and swimming patterns of the flagellates Paraphysomonas vestita and Pteridomonas danica

Most flagellates with hispid flagella, that is, flagella with rigid filamentous hairs (mastigonemes), swim in the direction of the flagellar wave propagation with an anterior position of the flagellum. Previous analysis was based on planar wave propagation showing that the mastigonemes pull fluid along the flagellar axis. In the present study, we investigate the flagellar motions and swimming patterns for two flagellates with hispid flagella: Paraphysomonas vestita and Pteridomonas danica. Studies were carried out using normal and high-speed video recording, and particles were added to visualize flow around cells generating feeding currents. When swimming or generating flow, P. vestita was able to pull fluid normal to, and not just along, the flagellum, implying the use of the mastigonemes in an as yet un-described way. When the flagellum made contact with food particles, it changed the flagellar waveform so that the particle was fanned towards the ingestion area, suggesting mechano-sensitivity of the mastigonemes. Pteridomonas danica was capable of more complex swimming than previously described for flagellated protists. This was associated with control of the flagellar beat as well as an ability to bend the plane of the flagellar waveform.     

Subpopulations of slowly cycling cells in S and G2 phase in mouse epidermis

Evidence has been presented supporting the existence of heterogeneity in cell-cycle progression in mouse epidermis, The present study was undertaken to characterize this heterogeneity in more detail. Hairless mice were continuously labelled with tritiated thymidine every 4 hr for 4 days. Basal cell suspensions were prepared from slices of mouse skin at intervals during the experiment and subjected to DNA flow cytometry. Cell-cycle analysis was combined with sorting of cells from windows in G1, S and G2 phase, and the proportion of labelled cells within each window was determined in autoradiographs. Reanalysis and resorting to control the purity of of sorted fractions were performed. Computer simulations of the data were made using a mathematical model assuming different S and G2 phase characteristics. A good fit to the data was only obtained when heterogeneity in mouse epidermal cell-cycle progression was assumed, indicating the existence of slowly traversing, distinct subpopulations of cells in G2 and S phase. These cells are assumed to contribute to about 40% of all cells in S phase and to about 70% of all in G2 phase. The estimated residence times in the resting states were 38 and 32 hr in S and G2 phase, respectively. Two-parameter sorting based on DNA and light scatter indicated that slowly cycling cells were larger than the average. There is no evidence of significant subpopulations of permanently non-proliferating keratinocytes in any of the cell-cycle phases.     

Abundance and Biomass of Heterotrophic Flagellates, and Factors Controlling Their Abundance and Distribution in Sediments of Botany Bay

The abundance and biomass of heterotrophic flagellates were estimated monthly in sediments of Botany Bay during March 1999-February 2000. The annual abundance and biomass were in the ranges of 0.46-4.70 x 10(5) cells/cm(3) and of 0.30-8.61 micro g C/cm(3), respectively. The majority of heterotrophic flagellates (93-100%) were less than 10 mm in length and few flagellates were larger than 10 mm. Of the total microbial carbon biomass, heterotrophic flagellates made up about 5% (but at times up to 35%). The contribution of heterotrophic flagellates varied from month to month, and among the sites. The abundance of heterotrophic flagellates was negatively correlated with sediment grain size and positively correlated with the abundance of bacteria, algae (autotrophic flagellates and diatoms), and their probable grazers. A best subsets regression analysis showed that bacterial and algal abundance are the most important factors controlling the abundance of heterotrophic flagellates. When the previously reported grazing rates on bacteria were applied, heterotrophic flagellates would consume a maximum of 64% of bacterial standing stock daily in Botany Bay, suggesting that heterotrophic flagellates are important as bacterivores. However, the importance of heterotrophic flagellate grazing probably varies significantly among the sites and from month to month.     

Plasmid Transfer between Marine Vibrio Strains during Predation by the Heterotrophic Microflagellate Cafeteria roenbergensis

The effect of grazing by the heterotrophic microflagellate Cafeteria roenbergensis on plasmid transfer between marine Vibrio S14 strains was studied by using artificial seawater. Several factors of potential importance for regulation of the plasmid transfer, such as nutrient release, production of a flagellate-derived substance(s) that may affect plasmid transfer, and the presence of surfaces, were investigated. Only living flagellates gave rise to, at instances, plasmid transfer enhanced more than 2 orders of magnitude. We propose that the activity of grazing flagellates allows for the significant increase in plasmid transfer observed under predation conditions. This may be due to a localized increase in bacterial numbers through filter feeding, thus providing high cell densities with increased possibility for cell-to-cell-contact and hence plasmid transfer.     

Mathematical model analysis of mouse epidermal cell kinetics measured by bivariate DNA/anti-bromodeoxyuridine flow cytometry and continuous [3H]-thymidine labelling

In a previous study the epidermal cell kinetics of hairless mice were investigated with bivariate DNA/anti-bromodeoxyuridine (BrdU) flow cytometry of isolated basal cells after BrdU pulse labelling. The results confirmed our previous observations of two kinetically distinct sub-populations in the G2 phase. However, the results also showed that almost all BrdU-positive cells had left S phase 6-12 h after pulse labelling, contradicting our previous assumption of a distinct, slowly cycling, major sub-population in S phase. The latter study was based on an experiment combining continuous tritiated thymidine [( 3H]TdR) labelling and cell sorting. The purpose of the present study was to use a mathematical model to analyse epidermal cell kinetics by simulating bivariate DNA/BrdU data in order to get more details about the kinetic organization and cell cycle parameter values. We also wanted to re-evaluate our assumption of slowly cycling cells in S phase. The mathematical model shows a good fit to the experimental BrdU data initiated either at 08.00 hours or 20.00 hours. Simultaneously, it was also possible to obtain a good fit to our previous continuous labelling data without including a sub-population of slowly cycling cells in S phase. This was achieved by improving the way in which the continuous [3H]TdR labelling was simulated. The presence of two distinct subpopulations in G2 phase was confirmed and a similar kinetic organization with rapidly and slowly cycling cells in G1 phase is suggested. The sizes of the slowly cycling fractions in G1 and G2 showed the same distinct circadian dependency. The model analysis indicates that a small fraction of BrdU labelled cells (3-5%) was arrested in G2 phase due to BrdU toxicity. This is insignificant compared with the total number of labelled cells and has a negligible effect on the average cell cycle data. However, it comprises 1/3 to 1/2 of the BrdU positive G2 cells after the pulse labelled cells have been distributed among the cell cycle compartments.     

Occurrence of mixotrophic flagellates in relation to bacterioplankton production, light regime and availability of inorganic nutrients in unproductive lakes with differing humic contents

1. Field data from five unproductive Swedish lakes were used to investigate the occurrence of mixotrophic flagellates in relation to bacterioplankton, autotrophic phytoplankton, heterotrophic flagellates and abiotic environmental factors. Three different sources of data were used: (i) a 3‐year study (1995–97) of the humic Lake Örträsket, (ii) seasonal measurements from five lakes with widely varying dissolved organic carbon (DOC) concentrations, and (iii) whole lake enrichment experiments with inorganic nutrients and organic carbon. 2. Mixotrophic flagellates usually dominated over autotrophic phytoplankton in Lake Örträsket in early summer, when both bacterial production and light levels were high. Comparative data from the five lakes demonstrated that the ratio between the biomasses of mixotrophic flagellates and autotrophic phytoplankton (the M/A‐ratio) was positively correlated to bacterioplankton production, but not to the light regime. Whole lake carbon addition (white sugar) increased bacterial biomass, and production, reduced the biomass of autotrophs by a factor of 16, and increased the M/A‐ratio from 0.03 to 3.4. Collectively, the results indicate that the dominance of mixotrophs among phytoplankton was positively related to bacterioplankton production. 3. Whole lake fertilisation with nitrogen (N) and phosphorus (P) demonstrated that the obligate autotrophic phytoplankton was limited by N. N‐addition increased the biomass of the autotrophic phytoplankton but had no effect on mixotrophic flagellates or bacteria, and the M/A‐ratio decreased from 1.2 to 0.6 after N‐enrichment. Therefore, we suggest that bacteria under natural conditions, by utilising allochthonous DOC as an energy and carbon source, are able to outcompete autotrophs for available inorganic nutrients. Consequently, mixotrophic flagellates can become the dominant phytoplankters when phagotrophy permits them to use nutrients stored in bacterial biomass. 4. In Lake Örträsket, the biomass of mixotrophs was usually higher than the biomass of heterotrophs during the summer. This dominance could not be explained by higher grazing rates among the mixotrophs. Instead, ratios between mixotrophic and heterotrophic biomass (the M/H‐ratio) were positively related to light availability. Therefore, we suggest that photosynthesis can enable mixotrophic flagellates to outcompete heterotrophic flagellates.     

Differences of the protozoan biomass and grazing during spring and summer in the Indian sector of the Southern Ocean

The dynamics of protozoa were investigated during two cruises in the Indian sector of the Southern Ocean: the early spring ANTARES 3 cruise (28 September to 8 November 1995) and the late summer ANTARES 2 cruise (6 February to 8 March 1994). Biomass and feeding activity of protozoa were measured as well as the biomass of their potential prey – bacteria and phototrophic flagellates – along the 62°E meridian. The sampling grid extended from the Polar Frontal region to the Coastal and Continental Shelf Zone in late summer and to the ice edge in spring, crossing the Antarctic Divergence. Protozoan biomass, although low in absolute terms, contributed 30% and 20% to the total microbial biomass (bacteria, phytoplankton and protozoa) in early spring and late summer, respectively. Nanoprotozoa dominated the total protozoan biomass. The geographical and seasonal distribution of protozoan biomass was correlated with that of phototrophic flagellates. However, bacterial and phototrophic flagellate biomass were inversely correlated. Phototrophic flagellates dominated in the Sea Ice Zone whereas bacteria were predominant at the end of summer in the Polar Frontal region and Coastal and Continental Shelf Zone. Furthermore, bacteria were the most important component of the microbial community (57% of the total microbial biomass) in late summer. Phototrophic flagellates were ingested by both nano-and microprotozoa. In contrast, bacteria were only ingested by nanoprotozoa. Protozoa controlled up to 90% of the daily bacterial production over the period examined. The spring daily protozoan ingestion controlled more than 100% of daily phototrophic flagellate production. This control was less strong at the end of summer when protozoan grazing controlled 42% of the daily phototrophic flagellate production. Accepted: 30 October 1999     

Fluorescence-activated cell sorting of specific affibody-displaying staphylococci

Efficient enrichment of staphylococcal cells displaying specific heterologous affinity ligands on their cell surfaces was demonstrated by using fluorescence-activated cell sorting. Using bacterial surface display of peptide or protein libraries for the purpose of combinatorial protein engineering has previously been investigated by using gram-negative bacteria. Here, the potential for using a gram-positive bacterium was evaluated by employing the well-established surface expression system for Staphylococcus carnosus. Staphylococcus aureus protein A domains with binding specificity to immunoglobulin G or engineered specificity for the G protein of human respiratory syncytial virus were expressed as surface display on S. carnosus cells. The surface accessibility and retained binding specificity of expressed proteins were demonstrated in whole-cell enzyme and flow cytometry assays. Also, affibody-expressing target cells could be sorted essentially quantitatively from a moderate excess of background cells in a single step by using a high-stringency sorting mode. Furthermore, in a simulated library selection experiment, a more-than-25,000-fold enrichment of target cells could be achieved through only two rounds of cell sorting and regrowth. The results obtained indicate that staphylococcal surface display of affibody libraries combined with fluoresence-activated cell sorting might indeed constitute an attractive alternative to existing technology platforms for affinity-based selections.     

The predatory soil flagellate Heteromita globosa stimulates toluene biodegradation by a Pseudomonas sp

A model food chain was established to investigate the influence of grazing by flagellates on bacteria degrading toluene in batch culture. The rate of toluene consumed by a Pseudomonas sp. strain PS+ (max. 0.37 fmol cell(-1) h(-1)) was significantly higher in the presence of the bacterivorous flagellate Heteromita globosa (max. 1.38 fmol cell(-1) h(-1)). A maximum increase of up to 7.5 times was observed in the rate of toluene consumed by these bacteria during exponential growth of this flagellate. Carbon conversion efficiency (CCE) of bacteria to flagellate biomass was estimated to be 33.4% based on measured biovolumes and published values for carbon contents. However, the CCE for toluene-derived carbon was lower (max. 4.9%) when calculations were based on incorporation of [ring-U-(14)C]toluene into biomass of flagellates grazing on labelled bacteria. The findings suggest a potential role for flagellates in bioremediation processes.     

Effects of Pulse Labelling With Tritiated Thymidine On the Circadian Rhythmicity In Epidermal Cell-Cycle Distribution In Mice

The influence of pulse labelling with 50 °Ci tritiated thymidine ([³H]TdR) (2 μCi/g) on epidermal cell-cycle distribution in mice was investigated. Animals were injected intraperitoneally with the radioactive tracer or with saline at 08.00 hours, and groups of animals were sacrificed at intervals during the following 32 hr. Epidermal basal cells were isolated from the back skin of the animals and prepared for DNA flow cytometry, and the proportions of cells in the S and G₂ phases of the cell cycle were estimated from the obtained DNA frequency distributions. the proportions of mitoses among basal cells were determined in histological sections from the same animals, as were the numbers of [³H]TdR-labelled cells per microscopic field by means of autoradiography. The results showed that the [³H]TdR activity did not affect the pattern of circadian rhythms in the proportions of cells in S, G₂ and M phase during the first 32 hr after the injection. the number of labelled cells per vision field was approximately doubled between 8 and 12 hr after tracer injection, indicating an unperturbed cell-cycle progression of the labelled cohort. In agreement with previous reports, an increase in the mitotic index was seen during the first 2 hr. These data are in agreement with the assumption that 50 °Ci [³H]TdR given as a pulse does not perturb cell-cycle progression in mouse epidermis in a way that invalidates percentage labelled mitosis (PLM) and double-labelling experiments.     

Structure and functional analysis of the microbial community in an aerobic: anaerobic sequencing batch reactor (SBR) with no phosphorus removal

The bacterial community of an aerobic:anaerobic non-P removing SBR biomass fed a mixture of acetate and glucose was analysed using several 16S rRNA based methods. Populations responsible for anaerobic glucose and acetate assimilation were determined with fluorescent in situ hybridization (FISH) in combination with microautoradiography (FISH/MAR). At 'steady state' this community consisted of alpha-Proteobacteria (26%) and gamma-Proteobacteria (14%), mainly appearing as large cocci in tetrads (i.e. typical 'G-Bacteria'). Large numbers of low G+C bacteria (22%), and high G+C Gram-positive bacteria (29%) seen as small cocci in clusters or in sheets were also detected after FISH. DGGE fingerprinting of PCR amplified 16S rDNA fragments and subsequent cloning and sequencing of several of the major bands led to the identification of some of these populations. They included an organism 98% similar in its 16S rRNA sequence to Micropruina glycogenica, and ca. 76% of the high G+C bacteria responded to a probe MIC 184, designed against it. The rest responded to the KSB 531 probe designed against a high G+C clone sequence, sbr-gs28 reported in other similar systems. FISH analyses showed that both these high G+C populations were almost totally dominated by small clustered cocci. Only ca. 2% of cells were beta-Proteobacteria. None of the alpha- and gamma-Proteobacterial 'G-bacteria' responded to FISH probes designed for the 'G-Bacteria' Amaricoccus spp. or Defluvicoccus vanus. FISH/MAR revealed that not all the alpha-Proteobacterial 'G-Bacteria' could take up acetate or glucose anaerobically. Almost all of the gamma-Proteobacterial 'G-Bacteria' assimilated acetate anaerobically but not glucose, the low G+C clustered cocci only took up glucose, whereas the high G+C bacteria including M. glycogenica and the sbr-gs28 clone assimilated both acetate and glucose. All bacteria other than the low G+C small cocci and a few of the alpha-Proteobacteria accumulated PHB. The low G+C bacteria showing anaerobic glucose assimilation ability were considered responsible for the lactic acid produced anaerobically by this SBR biomass, and M. glycogenica for its high glycogen content.     

Rapid, automated separation of specific bacteria from lake water and sewage by flow cytometry and cell sorting.

The use of fluorescence-activated flow cytometric cell sorting to obtain highly enriched populations of viable target bacteria was investigated. Preliminary studies employed mixtures of Staphylococcus aureus and Escherichia coli. Cells of S. aureus, when mixed in different proportions with E. coli, could be selectively recovered at a purity in excess of 90%. This was possible even when S. aureus composed only approximately 0.4% of the total cells. Cell sorting was also tested for the ability to recover E. coli from natural lake water populations and sewage. The environmental samples were challenged with fluorescently labelled antibodies specific for E. coli prior to cell sorting. Final sample purities of greater than 70% were routinely achieved, as determined by CFU. Populations of E. coli released into environmental samples were recovered at greater than 90% purity. The use of flow cytometry and cell sorting to detect and recover viable target bacteria present at levels of less than 1% within an indigenous microflora was also demonstrated.     

Fluorescence-Activated Cell Sorting of Specific Affibody-Displaying Staphylococci

Efficient enrichment of staphylococcal cells displaying specific heterologous affinity ligands on their cell surfaces was demonstrated by using fluorescence-activated cell sorting. Using bacterial surface display of peptide or protein libraries for the purpose of combinatorial protein engineering has previously been investigated by using gram-negative bacteria. Here, the potential for using a gram-positive bacterium was evaluated by employing the well-established surface expression system for Staphylococcus carnosus. Staphylococcus aureus protein A domains with binding specificity to immunoglobulin G or engineered specificity for the G protein of human respiratory syncytial virus were expressed as surface display on S. carnosus cells. The surface accessibility and retained binding specificity of expressed proteins were demonstrated in whole-cell enzyme and flow cytometry assays. Also, affibody-expressing target cells could be sorted essentially quantitatively from a moderate excess of background cells in a single step by using a high-stringency sorting mode. Furthermore, in a simulated library selection experiment, a more-than-25,000-fold enrichment of target cells could be achieved through only two rounds of cell sorting and regrowth. The results obtained indicate that staphylococcal surface display of affibody libraries combined with fluoresence-activated cell sorting might indeed constitute an attractive alternative to existing technology platforms for affinity-based selections.     

Cell kinetics in mouse epidermis studied by bivariate DNA/bromodeoxyuridine and DNA/keratin flow cytometry.

Hairless mice were injected intraperitoneally with bromodeoxyuridine (Brd-Urd). Basal cells were isolated from epidermis, fixed in 70% ethanol, and prepared for bivariate BrdUrd/DNA flow cytometric (FCM) analysis. Optimum detection of incorporated BrdUrd in DNA was obtained by combining pepsin digestion and acid denaturation. The cell loss was reduced to a minimum by using phosphate-buffered saline containing Ca2+ and Mg2+ to neutralize the acid. The percentage of cells in S phase and the average uptake of BrdUrd per labelled cell in eight consecutive windows throughout the S phase were measured after pulse labelling at intervals during a 24 h period. Furthermore, the cell cycle progression of a pulse-labelled cohort of cells was followed up to 96 h after BrdUrd injection. In general the results from both experiments were in good agreement with previous data from 3H-thymidine labelling studies. The percentage of cells in S phase was highest at night and lowest in the afternoon, whereas the average uptake of BrdUrd per labelled cell showed only minor circadian variations. There were no indications that BrdUrd significantly perturbed normal epidermal growth kinetics. A cell cycle time of about 36 h was observed for the labelled cohort. Indications of heterogeneity in traverse through G1 phase were found, and the existence of slowly cycling or temporarily resting cells in G2 phase was confirmed. There was, however, no evidence of a significant population of temporarily resting cells in the S phase. Bivariate DNA/keratin FCM analysis revealed a high purity of basal cells in the suspensions and indicated that the synthesis of the differentiation-keratin K10 was turned on only in G1 phase and after the last division.     

Subpopulations of Slowly Cycling Cells In S and G2 Phase In Mouse Epidermis

Evidence has been presented supporting the existence of heterogeneity in cell-cycle progression in mouse epidermis, the present study was undertaken to characterize this heterogeneity in more detail. Hairless mice were continuously labelled with tritiated thymidine every 4 hr for 4 days. Basal cell suspensions were prepared from slices of mouse skin at intervals during the experiment and subjected to DNA flow cytometry. Cell-cycle analysis was combined with sorting of cells from windows in G¹, S and G², phase, and the proportion of labelled cells within each window was determined in autoradiographs. Reanalysis and resorting to control the purity of sorted fractions were performed. Computer simulations of the data were made using a mathematical model assuming different S and G² phase characteristics. A good fit to the data was only obtained when heterogeneity in mouse epidermal cell-cycle progression was assumed, indicating the existence of slowly traversing, distinct subpopulations of cells in G² and S phase. These cells are assumed to contribute to about 40% of all cells in S phase and to about 70% of all in G² phase. the estimated residence times in the resting states were 38 and 32 hr in S and G² phase, respectively. Two-parameter sorting based on DNA and light scatter indicated that slowly cycling cells were larger than the average. There is no evidence of significant subpopulations of permanently non-proliferating keratinocytes in any of the cell-cycle phases.