Detection of mitogen induced stimulation of leukocytes from the rainbow trout (Oncorhynchus mykiss) by flow cytometric analysis of intracellular calcium

Flow cytometric analysis of the forward/side light scatter (FSC/SSC) of density gradient-separated head kidney cells of the rainbow trout revealed three distinctly separated populations, which we defined as population 1, 2 and 3. In spleen cells, populations 1 and 2 were also found, whereas population 3 was not detected. Further characterization regarding the surface Ig (sIg) revealed that population 2 of the head kidney and spleen contained 37.4 and 34.4% sIg⁺-cells, respectively. Incubation of the head kidney and spleen cells with different concentrations of concanavalin A (ConA), phytohemagglutinin (PHA) and [PWM] induced a pronounced intracellular calcium increase only in cells of population 2. This reaction was concentration dependent and caused by a release of intracellular Ca²⁺-stores. FMLP, a chemotactic peptide, had no effect on intracellular calcium response in all three populations. Similarly, the stimulation with PMA had no effect. This indicates that population 2 of the head kidney as well as the spleen is characterized by a high forward and low side light scatter and contains both subpopulation of lymphocytes, B- and T-cells. We demonstrated that the analysis of intracellular calcium increase due to mitogens is a suitable approach to identify lymphocytes in fish and enables further functional studies in these cells.     

A new method for fish leucocyte counting and partial differentiation by flow cytometry

A simple and rapid method for analysis of fish blood cells is presented. Carp (Cyprinus carpio) blood was diluted 200 times with Hanks' solution containing 1 microg/ml of DiOC6(3) which is a fluorescent, lipophilic dye. After staining for 10 min, the blood cells were measured by a flow cytometer (FACS). Several blood cell populations were identified by different FL-1 (green fluorescence), FSC (forward scatter), and SSC (side scatter) properties. FL-1 v. SSC or FSC v. SSC dot-plot of stained blood cells displayed five separate cell populations: erythrocytes: a mixture of thrombocytes plus lymphocytes; monocytes; neutrophils; and basophils. The number of each type of blood cell counted by the FACS was in good agreement with those counted microscopically.     

Measuring some flounder (Platichthys flesus L.) innate immune responses to be incorporated in effect biomonitoring concepts

For an implementation of innate immune responses of flounder (Platichthys flesus) in an integrated biological effect monitoring concept, leucocytes were isolated from peripheral blood, head kidney and spleen, and analysed for their capacity to mount a respiratory burst response upon phorbol ester stimulation. Responding cells were identified by reduced nitro-blue-tetrazolium salt deposits and by dihydro-rhodamine fluorescence in light microscope and flow cytometric analysis. Responding cells were found in head kidney derived cell suspensions rather than in peripheral blood or spleen. Parallel cytometric and microscopic analysis indicated that responding cells had a granulocyte or monocyte morphology, were alpha-naphtyl-esterase or myeloperoxidase positive and in flow cytometry exhibited a characteristic forward and side scatter (FSC/SSC) pattern. These cells represented 30–40% of head kidney derived cell suspensions and only 4–5 % of peripheral blood and spleen. In order to reduce sampling effort in field studies, leucocyte cell suspensions derived from flounder head kidney could be used in respiratory burst assays without further enrichment protocols. This paper combines, for the first time, conventional and cytometric analysis of phagocytes derived from flounder peripheral blood and head kidney. Communicated by H. v. Westernhagen, A. Diamant     

ISOLATION AND FLOW CYTOMETRIC CHARACTERIZATION OF PROTOPLASTS FROM MARINE MACROALGAE

Protoplasts were isolated from Ulva rigida C. Agardh (Chlorophyta) and two species of Rhodophyta , Gracilariopsis lemaneiformis ( Bory) Dawson, Acleto et Folvik and Gracilaria tenuistipitata Chang et Xia var . liui with minor modifications (the inclusion of 0.01% agarase in the set of cell-wall-degrading enzymes for the two red algae). Flow cytometric characteristics of freshly isolated protoplasts were determined on a FACScan flow cytometer (FC). The most useful parameters for characterizing protoplasts from marine algae were forward angle light scatter (FSC), orange fluorescence (FL2) and red fluorescence (FL3). Protoplasts from all the species were easily distinguishable when their FSC, FL2, and FL3 signals were combined in the bivariate plots FL3 vs. FSC and FL3 us. FL2. Two alternative techniques to help identify protoplasts from debris in the FC computer screen were developed (for FC without sorting capability). Both techniques were based on the ability of new FCs to record time. The first one was based on the induction of rapid changes of cell volume in response to osmotic stress. Only intact protoplasts responded to changes in the osmotic pressure. The second one was based on the uptake and hydrolysis of fluorescein diacetate by intracellular esterases. Viable protoplasts showed a hyperbolic accumulation of fluorescein with time. Semimaximal fluorescein accumulation was attained in 30.5 ± 9.5 s. Debris was easily recognized since, contrary to protoplasts, it did not show a time-dependent accumulation of fluorescein .     

Rapid detection of neutral lipid in green microalgae by flow cytometry in combination with Nile red staining—an improved technique

A staining protocol for rapid in situ detection of neutral lipid using flow cytometry in combination with Nile red staining was optimized. Staining efficiency was tested in terms of fluorescence intensity (% grandparent) in varied concentrations of Nile red and dimethyl sulfoxide (DMSO), with variable incubation period, temperature and pH level. The improved method was tested using two microalgae: Chlorella ellipsoidea and Chlorococcum infusionum. Maximum staining efficiency was recorded with a concentration of 5 μg mL−1 Nile red and 40 % DMSO in a 15-min incubation at 40 °C for both taxa (pH 6.5). The forward (FSC) and side scatter (SSC) two-dimensional dot plot showed highly scattered cells containing neutral lipid. The coefficient of variation, standard deviation, mean and median values were determined for quantification of neutral lipid. We also applied this modified method to detect the elevated level of neutral lipid in nitrate (NaNO3)-depleted cells; the efficiency of this technique was justified indicating a prominent 3- to 4-fold increase in neutral lipid in treated cells. Confocal images of stained cells also revealed accumulation of high levels of neutral lipid in treated microalgal cells.     

Micro-scale flow cytometry-based and biochemical analysis of lipid signaling in primary B cell subpopulations

B cell subpopulations in the spleen have been extensively characterized phenotypically; however, biochemical properties of these cell populations following B cell antigen receptor engagement have not been fully determined due to technical difficulties and limiting cell numbers. We therefore employed mini-scale protocols to assess lipid signaling, particularly that of diacylglycerol and inositol trisphosphate, with as few as 0.5×10⁶ purified early (T1) and late (T2) transitional B cells. Additionally, utilizing flow cytometric techniques, we determined levels of phosphatidylinositol bisphosphate and calcium mobilization in T1 and T2 cells, as well as mature follicular and marginal zone B cells using less than 1×10⁶ primary B cells. Thus, these biochemical and flow cytometric methodologies can be used to analyse signal-induced changes in phosphatidylinositol bisphosphate levels, diacylglycerol and inositol triphosphate production and calcium in each B cell population. These authors contributed equally.     

Changes in cell-surface expression of MHC and Thy-1.2 determinants following treatment with lipid modulating agents

Treatment of normal mouse spleen cells with lipid fluidity modulators changes the expression of cell-surface H-2 determinants. BALB/c spleen cells treated for 1 to 2 hr with cholesteryl hemisuccinate (CHS) displayed reduced levels of all tested H-2 determinants (H-2L, H-2K, and H-2D) as evaluated by flow microfluorometry and increased membrane lipid packing density as determined by 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence polarization. In contrast, decreasing membrane lipid packing density by phosphatidylcholine treatment decreased DPH fluorescence polarization and increased the expression of MHC determinants. The effects were selective in that expression of Thy-1.2 determinants was decreased by the latter treatment and not increased by CHS. The results are discussed in terms of passive modulation of antigenic expression.     

Steroid receptors in the developing and the adult rabbit endocervix and in endocervical epithelial cells isolated by flow cytometry

Immunocytochemistry with monoclonal antibodies H222 and JZB39 was used to study nuclear estrogen (ER) and progesterone (PgR) receptors, respectively, in the cervix during differentiation and in the adult rabbit. The undifferentiated state of the cervix of 2-week-old rabbits correlates with a paucity of immunoreactive nuclear ER, while the epithelium of most of these animals showed moderate immunostaining for the nuclear PgR. The cervical epithelium, stroma and muscle cells of 1-month-old rabbits, showed weak immunostaining for the ER, while staining for PgR remained comparable to that of 2-week-old rabbits. For 2-4-month old rabbits the epithelium was characterized by moderate immunostaining for the nuclear ER and strong immunostaining for the PgR. Strong, heterogeneous immunostaining for nuclear ER and PgR receptors in endocervical epithelial cells from 6-month-old (adult), estrous rabbits suggested there are subpopulations of cells that express differential sensitivity to steroid hormones. In order to characterize such subpopulations, live endocervical epithelial cells were sorted with a flow cytometer on the basis of forward angle light scatter (FSC) and side scatter (SSC) signals which correlated with cell size and secretory granule content, respectively. Secretory cells, as verified by ultrastructural analysis and histochemical staining, expressed the highest FSC and SSC signals and were designated fraction "a". Changes in the hormonal status of the animals altered the intrinsic light scatter properties of fraction "a" cells as follows: maximum FSC and SSC signals were reported for cells from estrous animals; ovariectomy or progesterone-dominance decreased cell size (FCS) and secretory granule content (SSC), while treatment of ovariectomized rabbits with estradiol increased both parameters. When fraction "a" cells from estrous rabbits were incubated with the monoclonal antibodies, two distinct subpopulations of secretory cells were identified by intensity and pattern of nuclear staining for the ER and PgR. Changes in the hormonal status of the animals produced changes in the intensity of nuclear immunostaining, however both cell types remained distinguishable on the basis of immunostain pattern reflecting either permanent or transitory differences in them, and differential hormone sensitivity. The presence of nuclear ER and PgR proteins in these cells confirms their function is bireceptor-mediated.     

Developmental changes in cyclin B1 and cyclin-dependent kinase 1 (CDK1) levels in the different populations of spermatogenic cells of the post-natal rat testis

Spermatogenesis is a highly ordered process which requires mitotic and meiotic divisions. In this work, we studied the relative changes in the levels of the two components of the M-phase promoting factor (MPF): the regulatory subunit cyclin B1 (CycB1) and its catalytic subunit cdk1, in spermatogenic cells of rats between 16 and 90 days of life. A multivariate flow cytometry analysis of forward scatter (FSC), side scatter (SSC) and DNA content was used to identify six populations of rat germ cells: spermatogonia with preleptotene spermatocytes, young pachytene spermatocytes, middle to late pachytene spermatocytes, secondary spermatocytes with doublets of round spermatids, round spermatids, and elongated spermatids. For any population studied no significant difference in the relative cellular content of CycB1 or cdk1 proteins between animals of different ages was observed. By contrast, CycB1 and cdk1 levels were different between the different populations of germ cells. CycB1 and cdk1 were rather high in young pachytene spermatocytes and culminated in late spermatocytes, i.e. just before the first meiotic division. The relative levels of the two proteins remained high in secondary spermatocytes then decreased in round spermatids at the exit of meiosis. Similar results were obtained by Western-blot analysis of total proteins obtained from lysates of elutriated fractions of spermatocytes and spermatids. MPF activity was assessed in lysates of germ cells from 32-day-old rats or adult animals using p13suc1 agarose and histone H1 as an exogenous substrate. H1 kinase activity was higher in pachytene spermatocytes than in round spermatid fractions from both adult and young rats. These results indicate that the meiotic G2/M transition is associated to high levels of CycB1 and cdk1 leading to high MPF activity irrespective of the age of the animals.     

Lipid synthesis: an indicator of antigen-induced signal transduction in antigen-binding cells.

A biochemical parameter of lymphocyte activation, lipid synthesis, has been measured in a purified specific antigen-binding cell population (ABC). ABC isolated form immune and nonimmune animals by sequential centrifugation on buoyant density and sedimentation velocity gradients have a 2- to 7-fold higher rate of 14-C choline incorporation into phospholipid than either unfractionated spleen cells or cells depleted of ABC. Aslo ABC from immune animals were shown to have a 4- to 7-fold higher rate of 14C-acetate incorporation into their neutral lipids than nonbinding controls. The elevated lipid synthesis seen in both nonimmune SRBC-ABC and TNP-SRBC ABC indicates that antigenic contact via the B cell immunoglobulin receptor results in signal transduction and activation of the specific receptor-bearing lymphocyte population. Binding of the same particle (SRBC) to B cells via their Fc receptors did not regularly result in activation of lipid synthesis. The magnitude of the increased lipid synthesis in ABC populations approached that seen in LPS-stimulated spleen cells. We propose that the measurement of early activation events in purified ABC may be a more appropriate criterion for antigen-induced signals that later events such as thymidine incorporation or antibody secretion.     

A unified procedure for conservative (morphology) and integral (DNA and immunophenotype) cell staining for flow cytometry

BACKGROUND: Current methods for multiparameter DNA flow cytometry suffer from several limitations. These include significant modifications of cell morphological parameters, the impossibility to counterstain cells with certain fluorochromes, and laborious tuning of the instrument that, for some procedures, must be equipped with an ultraviolet (UV) laser. To overcome these problems, we developed a novel method for the simultaneous analysis of morphological parameters, four-color immunophenotyping, and stoichiometric DNA labeling using a bench-top flow cytometer. METHODS: The method consists of a mild permeabilization/fixation treatment at room temperature, followed by labeling with fluorochrome-conjugated monoclonal antibodies (mAbs) and with the DNA dye 7-aminoactinomycin D (7-AAD) at 56 degrees C. RESULTS: Using this method, we analyzed resting peripheral blood mononucleated cells (PBMC), proliferating T cells cultured in the presence of interleukin-2 (IL-2), and lymphoblastoid B cells. Lymphocytes, monocytes, and lymphoblasts treated by this procedure retained differential light scattering (DLS) characteristics virtually identical to those of untreated cells. This allowed regions to be drawn on forward scatter (FSC) and side scatter (SSC) cytograms resolving different cell populations. DLS were preserved well enough to distinguish large lymphoblasts in the S or G2/M phases from small G0/G1 cells. Also, stainability with fluorescein-isothiocyanate (FITC), R-phycoerythrin (PE), allophycocyanin (APC)-conjugated mAbs was generally preserved. DNA labeling with 7-AAD was of quality good enough to permit accurate cell cycle analysis. CONCLUSIONS: The method described here, which we called integral hot staining (IHS), represents a very simple, reproducible, and conservative assay for multiparameter DNA analysis using a bench-top flow cytometer. Last but not least, the cytometer tuning for multiparameter acquisition is straightforward.     

Age-dependent and cell-population-restricted LRRK2 expression in normal mouse spleen

Leucine-rich repeat kinase 2 (LRRK2) is the causal molecule of familial Parkinson’s disease (PD), but its true physiological function remains unknown. In the normal mouse, LRRK2 is expressed in kidney, spleen, and lung at much higher levels than in brain, suggesting that LRRK2 may play an important role in these organs. Analysis of age-related changes in LRRK2 expression demonstrated that expression in kidney, lung, and various brain regions was constant throughout adult life. On the other hand, expression of both LRRK2 mRNA and protein decreased markedly in spleen in an age-dependent manner. Analysis of purified spleen cells indicated that B lymphocytes were the major population expressing LRRK2, and that T lymphocytes showed no expression. Consistently, the B lymphocyte surface marker CD19 exhibited an age-dependent decrease of mRNA expression in spleen. These results suggest a possibly novel function of LRRK2 in the immune system, especially in B lymphocytes.     

LPS regulation of the immune response: separate mechanisms for murine B cell activation by lipid A (direct) and polysaccharide (macrophage-dependent) derived from Bacteroides LPS

Lipopolysaccharide (LPS) derived from Bacteroides fragilis has been reported to stimulate mitogenic responses in spleen cell cultures from the classical LPS-hyporesponsive C3H/HeJ mouse strain; however, we have shown that purified splenic B cells from C3H/HeJ mice are hyporesponsive to phenol-water extracted LPS from B. fragilis ATCC 25285 (B-LPS). In the present study, B-LPS and its purified lipid A and polysaccharide components were tested for their ability to induce mitogenic and polyclonal IgM synthesis in spleen cell and purified splenic B cell cultures from classical LPS-responsive and -hyporesponsive mice. Mitogenic responses to B-LPS and E. coli K235 LPS(Ph) of whole spleen cells (2 X 10(5) cells/culture) or purified B cells (5 X 10(5) cells/culture) from classical LPS-responsive mouse strains (C3H/HeN, BALB/c, C57BL/6J, C57BL/10Sn, and DBA/2), F1 mice (derived from crosses between LPS responsive and C3H/HeJ mice), and classical LPS-hyporesponsive mice (C3H/HeJ and C57BL/10ScN) were high, intermediate, and low, respectively. When a higher number of whole spleen cells (5 X 10(5) cells/well) were cultured, B-LPS induced high mitogenic responses in C3H/HeN, intermediate responses in F1, and lower but significant responses in C3H/HeJ cultures. Similar results were obtained when polyclonal IgM synthesis was assessed in cultures containing 1 X 10(6) cells/culture. In contrast, the purified lipid A component of B-LPS failed to induce mitogenic responses in either whole spleen or purified B cell cultures. The addition of purified splenic B cells from C3H/HeJ mice to C3H/HeN or C3H/HeJ splenic adherent cells resulted in mitogenic responses to B-LPS, implying that the hyporesponsiveness to B-LPS seen in whole spleen cell cultures from C3H/HeJ mice at the lower cell concentration was due to limiting numbers of M phi. When splenic B cells and M phi from either C3H/HeN or C3H/HeJ mice were incubated with the lipid A or the polysaccharide moiety of B-LPS, lipid A induced mitogenic responses only in C3H/HeN cultures, whereas the polysaccharide moiety induced similar responses in both C3H/HeN and C3H/HeJ cultures. These results suggest that Bacteroides lipid A does not stimulate B cells from the classical LPS-hyporesponsive C3H/HeJ mouse strain, whereas the polysaccharide moiety of B-LPS is biologically active and mediates B cell stimulation via M phi.     

Separation of pigmented and albino melanocytes and the concomitant evaluation of endogenous peroxide content using flow cytometry

Flow cytometry (FCM) has been used extensively to analyze various biological properties of the cell. In this report, we describe a method by which FCM was used to determine the light scattering profile of a mixed population of pigmented and non-pigmented melanocytes, plus its subsequent use for the sorting and separation of the two cell types. In addition, the relative peroxide content in pigmented and non-pigmented melanocytes was compared by flow cytometry. Cultured avian melanocytes from a pigmented control and from three genetically distinct albino sources were studied. FCM analysis of forward versus side light scatter within a mixed suspension of pigmented and amelanotic melanocytes distinguished two overlapping populations of cells. Sorting of these two populations demonstrated that the population exhibiting much side and minimal forward light scatter was primarily pigmented melanocytes, while conversely the population exhibiting less side and more forward scatter was principally non-pigmented cells. These two melanocyte types also demonstrated differences in levels of endogenous peroxides. The intracellular content of peroxide in the two subpopulations of cells was measured utilizing the nonfluorescent compound, 2',7'-dichlorofluorescein diacetate (DCFH-DA), which within the cell is oxidized by intracellular peroxides to a fluorescent dichlorofluorescein (DCF). Non-pigmented albino melanocytes had the highest quantity of endogenous peroxides, while heavily pigmented cells had considerably less peroxide-related fluorescence. The amount of this DCF fluorescence could be enhanced by increasing concentrations of DCF used in the assay. These flow cytometric methods are useful for isolating and culturing subpopulations of melanocytes expressing various pigment levels and to investigate the relationship between melanin and its precursors with hydrogen and lipid peroxides in melanocytes.     

In vivo studies on the regeneration kinetics of enriched populations of haemopoietic spleen colony-forming cells from normal bone marrow

Functional properties of mouse haemopoietic spleen colony-forming cells, enriched 40- to 80-fold, from normal bone marrow were studied. It was found that: (1) the number of partially purified CFU-s (colony forming unit-spleen) required to rescue lethally irradiated mice was similar to the number of normal unfractionated bone marrow CFU-s giving the same level of protection; (2) the homing of partially purified CFU-s was similar to that of CFU-s from unfractionated bone marrow; (3) the regeneration of CFU-s in spleen was similar for enriched and unfractionated cell populations between 4 and 11 days after transplantation. In contrast, the rate of regeneration of CFU-s in femur was slower with enriched progenitor cells than with unfractionated bone marrow. The growth rate in femur, however, could be restored to normal by injecting freshly isolated syngeneic thymocytes with the enriched CFU-s population. The results indicate that the partially purified CFU-s are by themselves functionally normal and show that the rate of CFU-s repopulation in bone marrow can be affected by cell types other than spleen colony-forming cells.     

Flow Cytometric Characteristics of Sperm Cells Isolated from Pollen of Zea mays L

Sperm cells have been isolated from pollen of maize (Zea mays L.) and purified with Percoll density centrifugation. Their flow cytometric characteristics were determined on a FACScan flow cytometer with the fluorescent dyes, fluorescein diacetate and propidium iodide. Freshly isolated sperm cells appeared as a dot cluster on the forward scatter and side scatter dot plot. This dot cluster contained 85 to 95% of the 10 thousand counts collected. More than 98% of cells from the cluster were fluorescein diacetate positive, with no propidium iodide positivity, indicating high cell viability. After 5 hours in 15% (w/v) sucrose at room temperature (23°C), scattering properties, cell number, and percentage of fluorescein diacetate-positive cells remained the same. In contrast, Brewbaker and Kwack salts in 15% sucrose resulted in the emergence of a new cell population, as well as a decrease in cell number at 5 hours. Further investigations with individual components of the Brewbaker and Kwack salts showed that calcium was mainly responsible for the deleterious effects. These results demonstrate the utility of flow cytometry as a tool to determine viability and to monitor morphological changes of plant sperm cells and to challenge current views on the ability of Brewbaker and Kwack salts to maintain viability of isolated sperm cells.     

Changes in the lipid-accumulating cells of the liver during experimental hypervitaminosis A

Changes of the fat-storing cells (FSC) in the liver have been studied after oil solution of vitamin A have been injected to intact rats, when Kupffer's cells have been blocked with colloid silver and stimulated with prodigiosan. During these experiments (on the 4th, 7th, 14th days) the amount of FSC does not change, and their size increases. The blockade of Kupffer's cells does not influence the dynamics of the FSC changes, and their stimulation contributes to lipid accumulation in their cytoplasm and to earlier revealing of these elements (on the 2d day of the experiment). In parallel with increasing size of FSC, fatty infiltration of hepatocytes disappears and reticular fibers accumulate in the perisinusoid space. Participation of FSC in lipid metabolism and in synthesis of the III type collagen is proved, as well as existence of intercellular interactions of these elements with endotheliocytes, Kupffer's cells and hepatocytes.     

Internalization of surface HLA-DR molecules by human epidermal Langerhans cells: analysis by flow cytometry and confocal microscopy

Langerhans cells (LC) play a pivotal role in antigen processing and presentation to T cells during delayed-type hypersensitivity reaction in the skin. Antigen presentation involves the interaction between the class II molecules of MHC (HLA-DR) expressed by LC and T receptor of CD4⁺ T lymphocytes. It is now recognized that class II molecules are internalized into LC and can be associated with processed immunogenic peptides. This process involves receptor-mediated endocytosis. The aim of this study was to investigate the time-course of endocytosis of HLA-DR by freshly isolated human LC. Epidermal cells, obtained from normal skin samples, were labeled by indirect immunofluoresence using anti-HLA-DR monoclonal antibodies (MAb). The cell suspension was incubated at 37°C for different periods (15, 30, 45, 60 and 90 min) and then analyzed by flow cytometry and confocal microscopy. Flow cytometry analysis showed decreased HLA-DR molecule expression by LC after incubation at 37°C. Confocal microscopic analysis showed different strain patterns depending on the incubation time: (1)T=0, continuous peripheral staining; (2)T=15 min, patchy peripheral staining; (3)T=30 min, patches or intracellular vesicular staining; (4)T=45 min, intracellular vesicular staining; (5)T=60 min, diffuse intracellular staining; (6)T=90 min, aggregated staining. In our study model, flow cytometry provides quantitative information for the HLA-DR endocytosis, whereas confocal microscopy provides qualitative results concerning the intracellular distribution of internalized HLA-DR molecules. The use of the two complementary techniques allows us to characterize the spontaneous endocytosis of HLA-DR molecule by freshly isolated LC. Thisin vitro study model might be useful for testing the sensitizing potential of different chemical substances.Abbreviations Ab antibodies - APC antigen-presenting cells - BG Birbeck granules - DNCB 1-chloro-2,4-dinitrobenzene - DNFB 2,4-dinitrofluorobenzene - DTAF dichlorotriazinylfluorescein - FSC forward light scatter - LC Langerhans cells - LSCM laser scanning confocal microscopy - MHC major histocompatibility complex - MAb monoclonal antibodies - PFA paraformaldehyde - SSC side light scatter     

Optical compartmentation of vegetating algae species as a basis for their growth-specific characterization

BACKGROUND: The number of microalgal strains known to date is enormous and continuously growing, and their characterization accordingly requires quick and reliable methodologies. METHODS: Asynchronously growing logarithmic (3- and 6-day cultures) and stationary (9-day cultures) phase cell populations of two algae species that are difficult to distinguish microscopically (one Chlorella sp., C. vulgaris [c-27], and another that might belong to the same genus, SA-3 algae exsymbiotic from Paramecium bursaria) were characterized by means of flow cytometry (FCM). Forward light scatter (FSC) of algae was monitored in association with their 90 degrees side light scatter (SSC) and fluorescence of endogenous chlorophyll (FL3-height). RESULTS: Two-parameter FSC versus SSC and FSC versus FL3-height plots distinctly showed growth-specific compartmentation of algae into discrete cell subpopulations staying at a particular stage of the life cycle, and numbers of cells constituting these subpopulations could be quantitated. The growth pattern of C. vulgaris (c-27) differed substantially from that of SA-3 algae, particularly in the late-logarithmic (6-day) cultures. At this phase of growth, C. vulgaris (c-27) cells compartmentalized into three subpopulations, whereas SA-3 cells compartmentalized into two subpopulations. Different compartmentations of optical signals from late-logarithmic phase SA-3 algae and C. vulgaris (c-27) likely were caused by the differences in timing of the life cycle stages of these types of cells. CONCLUSIONS: Growth-specific compartmentation of vegetating microalgae by FCM provides a good basis for characterization of morphologically similar algae species. Because algae are also present in symbiotic relationships with other organisms, this tool might be of potential interest for the study of symbiosis mechanisms.