Microscopic Cell Counts Research Articles

RhoA activation stimulates IEC-6 cell proliferation by decreasing p21Wafl1/Cip1 and increasing Cdk2 expression in a polyamine dependent manner

Background & Aims: Although RhoA plays an important role m ceil proliferation and in Ras transformation in fibroblasts and mammary epithelial cells; its role in intestinal epithelial cells is still unknown. In our previous study, we showed that polyamine depletion (DFMO treatment) strongly inhibits proliferation of intestinal epithelial cells (IEC-6). In this report, we examined the effects of active RhoA and the mechanism underlying 1EC-6 cell proliferation, contact inhibition, and focus formation. We also examined whether polyamine depletion inhibits cell proliferation m the presence of constitutively active RhoA. Methods: Constitutively active RhoA and vector transfected 1EC-6 cell lines were used. Cells were grown in the presence or absence of DFMO, an irreversible inhibitor of omithine decarboxylase (ODC). Growth characteristics were assessed by microscopic observation, cell counting, and ceil cycle analysis by flow cytometry. Cell cycle regulators were evaluated by western blot, functional assay, and real-time PCR. Results: RhoA transfection significantly increased the rate of cell proliferation. These cells also demonstrated attenuated contact inhibition and conspicuous loci when they were fully confluent, increased proliferation was accompanied by decreased p21Wafl/Cipl, increased Cdk2 expression and activity. The inhibition of p21Wafl/Cipl was independent of p53. There was no activation of the Ras-Raf-Mek-Erk pathway in the RhoA transfected cell line. Polyamine depletion totally prevented the effect of activated RhoA on 1EC-6 cell proliferation and focus formation, decreased Cdk2 expression and activity independent of p21Wafl/Cipl. Conclusions: First, RhoA activation decreases p21, increase~ Cdk2 protein and Cdk2 activity, leading to the stimulation of intestinal epithelial cell proliferation and transformation; second, polyamine depletion totally prevents stimulating effect of RhoA on proliferation by decreasing Cdk2 expression and activity.

Effects of Light, Salinity and Inorganic Nitrogen on Cell Growth and Spirolide Production in the Marine Dinoflagellate Alexandrium ostenfeldii (Paulsen) Balech et Tangen

Small-volume batch cultures of the toxic marine dinoflagellate Alexandrium ostenfeldii (Paulsen) Balech et Tangen were exposed to gradients of salinity, light, and inorganic nitrogen to determine their effects on growth and toxin production. Cell growth was determined by in vivo fluorometry and microscopic cell counts, and spirolide production through the culture cycle was measured by liquid chromatography-mass spectrometry. Growth of A. ostenfeldii was a function of environmental conditions, and was strongly limited under sub-optimal regimes of light (≥ 150 pmol m - 2 s - 1 ), salinity (≤ 15 psu), and low nutrients (≤ 20 μM initial nitrate). Cell concentration and in vivo fluorescence were highly correlated (r 2 = 0.83 to 0.97, n = 6) but the relationship varied slightly depending on experimental conditions. Nevertheless, except under nitrogen limitation, fluorometry was an accurate method for tracking cell growth. The growth characteristics indicated that this estuarine isolate of A. ostenfeldii from Atlantic Canada was moderately shade-adapted, had a broad salinity tolerance, and could effectively utilize much higher levels of inorganic nitrogen than commonly occur in the natural environment. Analysis of toxin production and composition throughout the culture cycle showed that total concentration of spirolides in the cellular fraction of the culture (pmol l - 1 ) increased with cell concentration, but cell spirolide quota (Q s ) and toxin composition (% molar) did not vary substantially in response to variations in environmental regime. Spirolides accumulated in the growth medium, but the concentration remained low (< 15 % of total) in comparison to that in the cellular fraction. This study supports the conclusion that cell growth is directly responsive to changes in environmental variables, but spirolide production is constitutive and susceptible to acclimation in culture.

Selective in vivo growth of lymphocyte function- associated antigen-1-positive murine myeloma cells. Involvement of function-associated antigen-1-mediated homotypic cell-cell adhesion.

Lymphocyte function-associated antigen-1 (LFA-1) expression on multiple myeloma cells and its potential role in myeloma biology have been the subject of conflicting literature reports. In this study we used the 5T experimental mouse model to analyze the involvement of LFA-1 in myeloma cell bone marrow homing, survival, and growth. The 5T33MM vitro (5T33MMvt) myeloma line was used. LFA-1 and intercellular adhesion molecule-1 (ICAM-1) expression were analyzed by flow cytometry. A small molecule antagonist of LFA-1/ICAM interactions, BIRT 377, was used to block LFA-1 in vitro. Transendothelial migration was assessed by measuring migration through Transwells coated with bone marrow endothelial cells. Immediate in vivo homing was analyzed by tracing 51Cr-labeled cells. Invert microscopic cell counting was used to analyze homotypic cell adhesion. Cell cycle analysis was used to analyze apoptosis. S+G(2)/M phase analysis and 3H-thymidine incorporation were used to assess proliferation. Cells were separated into LFA-1(+) and LFA-1(-) fraction by magnetic activated cell sorting. 5T33MMvt cells had a heterogeneous LFA-1 expression and all cells were positive for the LFA-1 ligand ICAM-1. LFA-1 inhibition with BIRT 377 did not affect transendothelial migration of the 5T33MMvt cells; however, it did result in cell cluster scattering, indicating LFA-1 involvement in homotypic cell-cell adhesion. No effect was observed on apoptosis, but the percentage of cells in S+G(2)/M phase was decreased by 39%. 3H-thymidine incorporation confirmed this effect on 5T33MMvt cell proliferation (38% reduction). When 5T33MMvt cells were injected into animals, all myeloma cells isolated at the end stage of the disease were LFA-1(+) in contrast to the situation before injection. LFA-1(+) and LFA-1(-) MM cells had similar in vivo bone marrow homing capacities. Mice injected with LFA-1(+) 5T33MMvt cells developed myeloma (5/5) within 12 weeks after injection. In contrast, LFA-1(-) recipients did not develop the disease (0/5), even 1 year after tumor inoculation. Our data suggest that LFA-1-mediated homotypic cell-cell adhesion is involved in myeloma cell proliferation and raises the possibility that this interaction may have a crucial role in in vivo myeloma cell growth. LFA-1 does not appear to play a role in the bone marrow homing of these cells.

Bovine Mastitis in Zebu and Crossbred Cattle under the Extensive Management System in Tanzania

A study was carried out to evaluate the incidences and causes of bovine mastitis in Tanzanian shorthorn zebu (Bos indicus) in the traditional sector and crossbred cows (Bos taurus×Bos indicus) in the dairy ranching sector, both found under the extensive range management system. Management practices were evaluated through a survey study using structured questionnaires. A total of 120 lactating cows (60 cows from each sector) were screened for the disease using the California Mastitis Test (CMT). Confirmatory tests used for infected cows included; the Direct Microscopic Somatic Cell Count (DMSCC), culture, bacteriological and biochemical laboratory assays. Survey results showed that management practices were generally very poor in both sectors with 84% of the surveyed herds being kept and milked under very unhygienic environmental conditions. The level of infection was higher in the crossbred cows (5% clinical and 38.3% sub-clinical mastitis) and lower in the zebu cows with only sub-clinical mastitis (23.3%). Crossbred cows had (p<0.05) higher somatic cell counts than zebu cows. The four highest-ranking bacterial isolates in order of importance were Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae and Bacillus spp. It was concluded that bovine mastitis under the extensive management system in Tanzania was a result of poor management practices and that zebu cows were more resistant to the diseases than crossbred cows. (Asian-Aust. J. Anim. Sci. 2002. Vol 15, No. 5 : 751-756)

The new hematology analyzer Sysmex XE-2100: performance evaluation of a novel white blood cell differential technology.

Context The new hematology analyzer Sysmex XE-2100 (TOA Medical Electronics, Kobe, Japan) has a novel, combined, white blood cell differential technology and a special reagent system to enumerate nucleated red blood cells. Design Performance evaluation of both technologies of the Sysmex XE-2100 according to the H20-A protocol of the National Committee for Clinical and Laboratory Standards and comparison of the results with those for the hematology analyzer Sysmex NE-8000 (TOA Medical Electronics). Specimens Five hundred forty-four blood samples randomly chosen from various inpatient and outpatient departments of the Vienna University hospital. Results Five-part white blood cell differential counts on the XE-2100 revealed excellent correlation with the manual reference method for neutrophils, lymphocytes, and eosinophils (r =.925,.922, and.877, respectively) and good correlation for monocytes and basophils (r =.756 and.763, respectively). The efficiency rates of flagging for the presence of >/=1% abnormal white blood cells were 83% (XE-2100) and 66% (NE-8000). The correlation of automated and microscopic nucleated red blood cell counts was excellent (r =.97). Conclusions From the present evaluation and our former experience with other types of Sysmex analyzers, we conclude that the new white blood cell differential technology of the XE-2100 represents a further development toward more efficient flagging of abnormal white blood cells.

Using light scatter signal to estimate bacterial biovolume by flow cytometry

In the past decade, flow cytometry has become a useful and precise alternative to microscopic bacterial cell counts in aquatic samples. However, little evidence of its usefulness for the evaluation of bacterial biovolumes has emerged in from the literature. The light scattering and cell volume of starved bacterial strains and natural bacterial communities from the Black Sea were measured by flow cytometry and epifluorescence microscopy, respectively, in order to establish a relationship between light scattering and cell volume. With the arc-lamp flow cytometer, forward angle light scatter (FALS) was related to cell size in both the starved strains and natural communities, although regression parameters differed. We tested the predictive capacity of the FALS verous cell size relationship in a bacterial community from the North Sea. That analysis showed that a reliable bacterial biovolume prediction of a natural bacterial community can be obtained from FALS using a model generated from natural bacterial community data. Bacterial biovolume is likely to be related to FALS measurements. It is possible to establish a generally applicable model derived from natural bacterial assemblages for flow cytometric estimation of bacterial biovolumes by light scatter.

Effect of red blood cells on the antitumor activity of oxycellulose.

The antitumor activity of oxycellulose to MH134 tumor cells, evaluated with microscopic viable cell count, was suppressed by murine red blood cells (MRBCs) in a dose-dependent manner. The antitumor activity of oxycellulose was suppressed by MRBCs also when evaluated with 3H-TdR uptake. However, that of mitomycin C or 5-fluorouracil was not suppressed by MRBCs. On the other hand, the antitumor activity of oxycellulose was suppressed by MRBCs also when evaluated with 51Cr release assay. At that time, the antitumor activity of cisplatin to MH134 tumor cells were not suppressed by MRBCs. The antitumor activity of oxycellulose to MH134 tumor cells was suppressed by the membrane or the cytosol fraction of the lysate of MRBCs as well as intact MRBCs. When heated at 56 degrees C for 30 min, MRBCs were unable to suppress the activity of oxycellulose. It was examined whether or not proteins related to MRBCs (hemoglobin, catalase, superoxide dismutase or taurine) suppressed the activity of oxycellulose. They did not suppress the activity of oxycellulose.

Inverse relationship between Leydig cell density and metastatic potential of prostatic adenocarcinoma.

Purpose: Evaluate the relationship between metastatic potential of prostatic adenocarcinoma (PC) and testicular Leydig cell density. Materials and methods: Tissue samples from 111 men, age 52–85, with PC and bilateral orchiectomy were evaluated for Leydig cell density. The patients were divided into two groups: Group A were patients with metastasis (n=36) and Group B were patients without metastasis (n=75). Leydig cell density was determined by direct manual microscopic cell count on the tissue sections. The means of cell counts by four pathologists, expressed as cell/0.78 mm were used for analysis. The normally distributed data were analyzed by two‐tail Student’s t‐test. Thirty‐eight age‐compatible autopsy cases who died of unrelated causes served as normal controls. Results: The mean of Leydig cell count in group A patients was 14.43 (14.43 ± 1.19 SE). Mean of Group B was 47.05 (47.05 ± 4.05 SE) whereas normal controls displayed a mean of 48.66 (48.66 ± 2.94 SE). Group A was significantly different from control (p<0.00001). Group A and Group B were also significant different (p<0.001) whereas control was not significantly different from Group B (p>0.75). Conclusions: Patients with metastatic adenocarcinoma of prostate, as a group, have a significantly lower Leydig cell density than patients without metastasis or patients without PC in compatible age groups. The hormonal relationship between this observation is however unknown. One possible explanation is that PC subpopulation with metastatic potential may require different level of endogenous androgen or are androgen‐independent.

Quality assessment of protected specimen brush samples by microscopic cell count.

Protected specimen brushing (PSB), combined with quantitative culture, is now recognized as one of the reference methods for diagnosis of nosocomial pneumonia. However, no criteria exist with which to assess the quality of the PSB sample. We studied numbers of inflammatory cells and bronchial cells per microscopic field (magnification: x500, objective x50) in cytospin preparations of PSB samples. Results of cell count and quantitative culture in a first study period were compared with those in a second study period, following adaptation of the PSB technique and collection of samples from more peripheral sites. The cellular content of samples from patients and controls was investigated. We examined 86 samples from patients with suspected nosocomial pneumonia and 15 samples from uninfected controls. The number of samples with a high cellular content was considerably greater in the second study period. No positive cultures were obtained from samples containing < 10 cells per field. The numbers of cells in samples from uninfected controls were comparable to the numbers in samples from patients. Our results indicate that absence of cells probably represents inadequate sampling. Negative PSB cultures with cytospin preparations containing < 10 cells per microscopic field should therefore be considered with caution, and resampling considered.

LIGHT HISTORY INFLUENCES THE SENSITIVITY TO ATRAZINE IN PERIPHYTIC ALGAE

This study examines the relationship between light history (i.e. light conditions during colonization) and sensitivity to atrazine for periphyton communities grown on etched glass substrates (colonized for 2–3 weeks) at seven stream and river sites differing in water chemistry and light regime. For each site, taxonomic composition of the community (by microscopic examination and cell counts), pigment composition, and photosynthetic parameters were measured and related to sensitivity to the herbicide. Photosynthetic parameters were estimated from photosynthesis– irradiance (P‐I) curves; the responses of the algae to atrazine were analyzed using short‐term concentration–response tests. Periphyton communities colonizing shaded sites were diatom dominated, had higher percentages of accessory pigments, and were more susceptible to photoinhibition at high light intensities compared to periphyton communities colonizing open sites; communities in the more open sites were dominated by green algae or cyanobacteria. Periphyton communities colonizing open sites were more sensitive to atrazine; the concentration of atrazine needed to inhibit photosynthesis by 50% in the short‐term tests (EC50 values) was correlated with light conditions during colonization. The interaction between treatment factors (light conditions during colonization, irradiance of incubation, and atrazine concentration) was analyzed by three‐way ANOVA. The two‐way interactions were significant, indicating that the response of the periphyton to irradiance during incubation and to atrazine concentration both depend on light history. However, the magnitude of the interaction between light history and atrazine toxicity was attenuated by changes in irradiance during incubation (three‐way ANOVA). This investigation presents evidence that the apparent toxicity of atrazine to lotic periphyton in natural communities is greater for algae that are adapted to high‐light conditions than for those that are adapted to low‐light conditions.

Factors limiting microbial growth and activity at a proposed high-level nuclear repository, yucca mountain, nevada

As part of the characterization of Yucca Mountain, Nev., as a potential repository for high-level nuclear waste, volcanic tuff was analyzed for microbial abundance and activity. Tuff was collected aseptically from nine sites along a tunnel in Yucca Mountain. Microbial abundance was generally low: direct microscopic cell counts were near detection limits at all sites (3.2 x 10(sup4) to 2.0 x 10(sup5) cells g(sup-1) [dry weight]); plate counts of aerobic heterotrophs ranged from 1.0 x 10(sup1) to 3.2 x 10(sup3) CFU g(sup-1) (dry weight). Phospholipid fatty acid concentrations (0.1 to 3.7 pmol g(sup-1)) also indicated low microbial biomasses; diglyceride fatty acid concentrations, indicative of dead cells, were in a similar range (0.2 to 2.3 pmol g(sup-1)). Potential microbial activity was quantified as (sup14)CO(inf2) production in microcosms containing radiolabeled substrates (glucose, acetate, and glutamic acid); amendments with water and nutrient solutions (N and P) were used to test factors potentially limiting this activity. Similarly, the potential for microbial growth and the factors limiting growth were determined by performing plate counts before and after incubating volcanic tuff samples for 24 h under various conditions: ambient moisture, water-amended, and amended with various nutrient solutions (N, P, and organic C). A high potential for microbial activity was demonstrated by high rates of substrate mineralization (as much as 70% of added organic C in 3 weeks). Water was the major limiting factor to growth and microbial activity, while amendments with N and P resulted in little further stimulation. Organic C amendments stimulated growth more than water alone.

Tumor cell counting using an image analysis program for MIB-1 immunohistochemistry.

A cell counting method for MIB-1 immunohistochemistry, using an image analysis program, NIH Image, on a personal computer, has advantages over manual cell counting by microscopy. MIB-1 slides were photographed at a magnification of x 50 on 24 x 36 mm color films and the photographs were then enlarged to adequate size for observation. The MIB-1-positive cells, on the enlargements, were marked with a white pen, and negative cells with a black pen. Subsequently, the image of an enlargement was converted into data that can be processed by the program using a flatbed image scanner. Threshold levels were adjusted according to the respective white and black markings, and the program automatically counted the number of MIB-1-positive and negative tumor cells. Our method has the following significant advantages: Once cells had been marked and the image converted into the NIH Image data, the cell count process was completed within a few minutes; in contrast to manual microscopic cell counting, count overlapping or cells missed by the observer are eliminated; overlapping nuclei could be separated by the marking procedure; our method required no elaborate and costly image analysis system. The present method may be recommended to researchers undertaking cell kinetic studies utilizing immunohistochemical methods.

Annual profiles of photosynthetic lipophilic pigments in four freshwater lakes in relation to phytoplankton counts as well as to nutrient data

Phytoplankton structure and dynamics of four eutrophic lakes of Berlin were studied in 1992. The biomass change of relevant classes - Cyanophyceae, Bacillariophyceae, Cryptophyceae, and Chlorophyceae - is well represented by HPLC-analysis of photosynthetic lipophilic pigments simultaneously carried-out. Nevertheless, an estimation of algal class biovolumes or phytoplankton composition, on the basis of chromatographically recorded pigment data is restricted to a semi-quantitative consideration. Different pigment amounts in individual species of the same algal class or varying marker pigment/chlorophyll-a ratios are discussed as a reason for the misleading pigment-based quantification of phytoplankton dynamics. However, the analysis of the obtained data points to the possibility of using HPLC-aided pigment determinations in connection with microscopic cell counting for a detection of variable physiological states under field conditions; provided that mass developments of algae are dominated by a single species as common in eutrophic lakes. For time periods with dominating Planktothrix agardhii or Microcystis spec. a positive correlation was found between their content of the cyanophyte specific pigment echinenone and increasing nitrogen availability. In contrast, no relations could be detected between the changing fucoxanthin content of Aulacoseira spec. and any of the measured environmental parameters (nutrient concentrations or light climate).

Competition between Pseudomonas fluorescens Ag1 and Alcaligenes eutrophus JMP134 (pJP4) during colonization of barley roots

To use deliberately released beneficial microorganisms in the rhizosphere, we need a better understanding of the process of root colonization by seed-borne or soil-borne inocula. In this study, we determine the survival of Pseudomonas fluorescens Ag1 and Alcaligenes eutrophus JMP134, their colonization ability as affected by substrates, and the relative importance of migration versus competition for colonization of the root. Ag1 and the 2,4-dichlorophenoxy-acetic acid (2,4-D) degrader JMP134 were inoculated in sterile barley rhizosphere systems. After inoculation of seeds with individual strains, comparable population sizes were established in the rhizosphere as determined by immunofluorescence microscopic total cell counts. Both strains were motile and able to colonize the entire root system without percolating water to stimulate passive transport. Comparing immunofluorescence microscopic cell counts with colony-forming units demonstrated that a subpopulation of A. eutrophus JMP134 closely associated with the root was non-culturable in contrast to the population in rhizosphere soil. Hence, the sole use of culture-dependent methods may give misleading information about the distribution of bacteria in the rhizosphere. Colonization studies with both strains showed that co-inoculation of Ag1 and JMP134 caused a decrease of the population size of JMP134 if 2,4-D was not added to the soil as a specific carbon source for this strain. Thus, competition for limited carbon sources might influence the composition of the bacterial community in the rhizosphere. We also found that the presence of an established inoculum in the soil reduced subsequent root colonization by a seed-inoculated strain, probably by filling available niches, also indicating that competition from other bacteria may be an important factor determining the distribution of seed-borne inocula. This factor may be just as important for the distribution of bacteria as migration.

Competition between Pseudomonas fluorescens Ag1 and Alcaligenes eutrophus JMP134 (pJP4) during colonization of barley roots

To use deliberately released beneficial microorganisms in the rhizosphere, we need a better understanding of the process of root colonization by seed-borne or soil-borne inocula. In this study, we determine the survival of Pseudomonas fluorescens Ag1 and Alcaligenes eutrophus JMP134, their colonization ability as affected by substrates, and the relative importance of migration versus competition for colonization of the root. Ag1 and the 2,4-dichlorophenoxy-acetic acid (2,4-D) degrader JMP134 were inoculated in sterile barley rhizosphere systems. After inoculation of seeds with individual strains, comparable population sizes were established in the rhizosphere as determined by immunofluorescence microscopic total cell counts. Both strains were motile and able to colonize the entire root system without percolating water to stimulate passive transport. Comparing immunofluorescence microscopic cell counts with colony-forming units demonstrated that a subpopulation of A. eutrophus JMP134 closely associated with the root was non-culturable in contrast to the population in rhizosphere soil. Hence, the sole use of culture-dependent methods may give misleading information about the distribution of bacteria in the rhizosphere. Colonization studies with both strains showed that co-inoculation of Ag1 and JMP134 caused a decrease of the population size of JMP134 if 2,4-D was not added to the soil as a specific carbon source for this strain. Thus, competition for limited carbon sources might influence the composition of the bacterial community in the rhizosphere. We also found that the presence of an established inoculum in the soil reduced subsequent root colonization by a seed-inoculated strain, probably by filling available niches, also indicating that competition from other bacteria may be an important factor determining the distribution of seed-borne inocula. This factor may be just as important for the distribution of bacteria as migration.

Stability of chitosan gel as entrapment matrix of viable Scenedesmus bicellularis cells immobilized on screens for tertiary treatment of wastewater

An experiment with immobilized microalgal cells was carried out using high and low viscosity chitosan and konjac flour to enhance the stability of hardened gels during tertiary treatment of wastewaters containing a high concentration of phosphate salts (1 m Na 3PO 4). Mechanical properties as well as compression testing, moisture retention in gels and the uptake of nutrients, such as nitrogen and phosphorus, from artificial wastewater were studied in the immobilized gel state. The disruption due to chemical interactions of pure high and low viscosity chitosan or mixed gels with konjac flour was examined daily by microscopic counting of the total cells released in the medium during seven weeks. The removal of NH + 4-N and PO 3− 4-P was monitored during four nutrient starvation-removal cycles. A complete cycle consisted of starving immobilized cells for 48 h in air saturated at 100% relative humidity, followed by removal of nutrients from wastewater. The immobilization obtained from 2% (m/v) high viscosity chitosan gels showed a more significant chemical stability ( P ≤ 0.05) in medium with 1 m Na 3PO 4 that contained viable cells than low viscosity chitosan or mixed gels. Sodium pyrophosphate was the best chelating agent used for cross-linking and did not affect the diffusion of inorganic nutrients through the hardened gels or the microalgal growth inside the network of chitosan immobilized cells. After the second uptake, the elimination of NH + 4-N and PO 3− 4-P was 100% within 3 h, whereas after the fourth uptake, the nitrogen disappearance was 99% within 2 h and 100% for phosphorus with the same incubation time. After more than one month incubation of chitosan gel beads containing Scenedesmus bicellularis cells that had been soaked daily in a fresh 1 m Na 3PO 4 solution, the stability of hardened gels was not affected and no significant release ( P ≥ 0.05) of entrapped cells was observed in the medium.

Use of fluorochromes in the determination of chemotaxis and haptotaxis of granulocytes by micropore filter assays

Most conventional assays for the in vitro measurement of polymorphonuclear leukocyte (PMN) migration are modifications of the Boyden chamber technique, which requires quantification of migrated cells on micropore filters. This quantification is accompanied by several disadvantages, comprising errors in direct cell counting, low sensitivity of turbidimetric methods, the loss of marker enzymes by activation of PMNs, or the use of radioactive isotopes. We set up an improved fluorometric method to measure chemotactic and haptotactic migration of PMNs using polycarbonate filter-bearing Transwell culture plate inserts. This improved fluorometric method allows the evaluation of effects of fluorescent-dye labeling on chemotaxis and haptotaxis - defined as cell migration due to cell surface- or matrix-bound gradients of chemoattractants - by collecting data in an automated system. Results were compared with data obtained from direct microscopic cell counting in Transwell experiments as well as in conventional 48-multiwell Boyden chamber assays. Calcein-AM and BCECF-AM proved to be fluorochromes with minimal effect on both types of PMN migration. We conclude that the fluorochromes are powerful tools for the analysis of PMN migration and allow modifications of chemotaxis/haptotaxis assays for automated quantification.

The Impact on Tissue Utilization of Screening Donor Corneas by Specular Microscopy at the University of Texas Southwestern Medical Center

To determine how frequently specular microscopy results affect the outcome of eye-bank judgments on the transplantability of donor corneas, 1,011 consecutive donor records from a 3-year period at the Transplant Services Center of the University of Texas Southwestern Medical Center at Dallas were analyzed. Specular microscopy cell counts from each decade of donor age were determined, and it was found that there were no cell counts < 2,000 mm2 for any donor age < 40. Above age 40, the percentage of cell counts < 2,000 per mm2 rose from 3.9% for donors in their forties of 6% for donors in their seventies. For donors between 40 and 69 years, specular microscopy was used to rule out unacceptable tissue in an additional 3.3% of a prescreened pool of corneas evaluated by current Eye Bank Association of America standards. While corneas from donors over age 69 were initially presumed to be unacceptable for transplant at this eye bank, routine specular microscopic examination helped to clear for transplant of 31 corneas from donors of this age group.

A protective action of chondroitin sulfate proteoglycans against neuronal cell death induced by glutamate

The role of chondroitin sulfate proteoglycans (CSPGs) on excitotoxic cell death and long-term survival of neurons were investigated in primary cultured neurons of the rat cortex. Soluble CSPGs were prepared from 10-day-old and adult rat brains by the ion-exchange chromatography on DEAE-Sephacel. CSPGs were added to the culture medium on culture day 4, and glutamate neurotoxicity was examined on culture day 7 by both microscopic cell count and measurement of lactate dehydrogenase activity in culture media. The effect on long-term survival was evaluated by counting viable neurons until culture day 28. CSPGs and core proteins, but not glycosaminoglycan chains (GAGs), protected cultured neurons from excitotoxic cell death induced by 24 h exposure to 1 mM glutamate, but CSPGs did not promote the long-term survival of neurons. The neuroprotective effect of CSPGs and core proteins was dose-dependent with ED 50 about 10 μM hexuronate and 2 μg/ml protein respectively. This effect was not considered to be due to adsorption of glutamate by CSPGs because [ 3H]glutamate was not adsorbed by CSPGs added to the culture medium. Based on these findings, we suggested that CSPGs may exert their neuroprotective action through molecular interactions with the binding sites on neuronal membrane, neurotrophic factors, or other extracellular matrix molecules and may be involved in the pathogenesis of neuronal cell death in acute pathological conditions and chronic degenerative diseases of the brain.

Chapter 16 Reticulocyte Analysis and Reticulocyte Maturity Index

The traditional laboratory methodology of reticulocyte analysis for the past century has been the manual microscopic counting of erythrocytes or red cells on supravitally stained blood samples. Although slide-based methods, such as new methylene blue staining, do provide clinically useful information, the lack of precision in these subjective assays is well documented. In particular, the poor precision of manual counting methods does not allow for clinically useful sequential monitoring of patients with low reticulocyte levels or for therapeutic monitoring of drug therapies for either erythropoietic inhibition or enhancement. Methods of flow cytometric reticulocyte analysis for both clinical and experimental applications have been reported over the past years utilizing a variety of fluorescent dyes. Thiazole orange (TO) has distinct advantages over other nucleic acid binding dyes for reticulocyte analysis using commercial multipurpose flow cytometry (FCM) instruments and is currently utilized by the vast majority of clinical laboratories. TO reticulocyte analysis offers the ability to use easily reticulocyte enumeration and a stable, reproducible fluorescence intensity measurement over a 60-min period. Furthermore, the fluorescence signal provides a high quantum yield or good separation of the reticulocyte (positive) from the mature red blood cell (negative) populations.