A novel flow cytometric assay for measurement of In Vivo pulmonary neutrophil phagocytosis

Background  

Phagocytosis assays are traditionally performed in vitro using polymorphonuclear leukocytes (PMNs) isolated from peripheral blood or the peritoneum and heat-killed, pre-opsonized organisms. These assays may not adequately mimic the environment within the infected lung. Our laboratory therefore has developed a flow cytometric in vivo phagocytosis assay that enables quantification of PMN phagocytosis of viable bacteria within the lungs of rats. In these studies, rats are injected transtracheally with lipopolysaccharide (LPS) to recruit PMNs to their lungs. They are then infected with live 5(-and 6) carboxyfluorescein diacetate succinimidyl ester (CFDA/SE) labeled type 3 Streptococcus pneumoniae. Bronchoalveolar lavage is performed and resident alveolar macrophages and recruited PMNs are labeled with monoclonal antibodies specific for surface epitopes on each cell type. Three color flow cytometry is utilized to identify the cell types, quantify recruitment, and determine uptake of the labeled bacteria.     

Quantitative flow cytometric analysis of ABO red cell antigens.

A flow cytometry method has been employed to quantitatively compare the expression of A, B and H antigens on various red blood cells (RBC). The H substance was directly labelled by fluorescein-conjugated anti-H lectin and the A and B antigens by indirect staining first with monoclonal anti-A or anti-B antibodies followed by fluorescently, fluorescein (FITC) or phycoerythrin (PE), labelled anti-mouse immunoglobulin (Ig) antibodies. More than a ten-fold difference in cellular fluorescence intensity was found within each sample. Both the percentage and the mean fluorescence of the positive subpopulation for each antigen were determined. Each RBC population was characterized with respect to the expression of A, B or H antigen by a compound mean value that was the calculated product of these two parameters. The results demonstrated a reciprocal relationship between the compound means of A or B and H. The ratio of A/H or B/H was found to be most informative. Homozygotes for A or B had ratios of greater than 200 and greater than 30, respectively, while heterozygotes (AO or BO) had ratios of less than 5. This method could also distinguish between A1 and A2; RBC carrying the A1 phenotype (as determined by agglutination with anti-A1 lectin) showed a higher A/H ratio than those carrying A2. In contrast to the reciprocity in the expression of A (or B) and H found in RBC obtained from different individuals, a direct correlation was found in the expression of these antigens by individual cells within a given population.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative measurement of binding kinetics in sandwich assay using a fluorescence detection fiber-optic biosensor

Fiber-optic biosensors have been studied intensively because they are very useful and important tools for monitoring biomolecular interactions. Here we describe a fluorescence detection fiber-optic biosensor (FD-FOB) using a sandwich assay to detect antibody-antigen interaction. In addition, the quantitative measurement of binding kinetics, including the association and dissociation rate constants for immunoglobulin G (IgG)/anti-mouse IgG, is achieved, indicating 0.38 × 10⁶ M⁻¹ s⁻¹ for k_a and 3.15 × 10⁻³ s⁻¹ for k_d. These constants are calculated from the fluorescence signals detected on fiber surface only where the excited evanescent wave can be generated. Thus, a confined fluorescence-detecting region is achieved to specifically determine the binding kinetics at the vicinity of the interface between sensing materials and uncladded fiber surface. With this FD-FOB, the mathematical deduction and experimental verification of the binding kinetics in a sandwich immunoassay provide a theoretical basis for measuring rate constants and equilibrium dissociation constants. A further measurement to study the interaction between human heart-type fatty acid-binding protein and its antibody gave the calculated kinetic constants k_a, k_d, and K_D as 8.48 × 10⁵ M⁻¹ s⁻¹, 1.7 × 10⁻³ s⁻¹, and 2.0 nM, respectively. Our study is the first attempt to establish a theoretical basis for the florescence-sensitive immunoassay using a sandwich format. Moreover, we demonstrate that the FD-FOB as a high-throughput biosensor can provide an alternative to the chip-based biosensors to study real-time biomolecular interaction.     

Age-induced reprogramming of mast cell degranulation

Mast cell degranulation can initiate an acute inflammatory response and contribute to the progression of chronic diseases. Alteration in the cellular programs that determine the requirement for mast cell degranulation would therefore have the potential to dramatically impact disease severity. Mast cells are exposed to increased levels of PGE2 during inflammation. We show that although PGE2 does not trigger the degranulation of dermal mast cells of young animals, in older mice, PGE2 is a potent mast cell stimulator. Intradermal administration of PGE2 leads to an EP3 receptor-dependent degranulation of mast cells, with the number of degranulated cells approaching levels observed in IgE- and Ag-treated controls. Taken together, these studies suggest that the ability of PGE2 to initiate mast cell degranulation changes in the aging animal. Therefore, elevated PGE2 levels might provide an important pathway by which mast cells are engaged to participate in inflammatory responses in the elderly patient.     

A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6

ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.     

Quantitative determination of gap junction intercellular communication using flow cytometric measurement of fluorescent dye transfer

Gap junction intercellular communication (GJIC) is involved in several aspects of normal cell behaviour, and disturbances in this type of communication have been associated with many pathological conditions. Reliable and accurate methods for the determination of GJIC are therefore important in studies of cell biology. (Tomasetto, C., Neveu, M.J., Daley, J., Horan, P.K. and Sager, R. (1993) Journal of Cell Biology, 122, 157-167) reported some years ago the use of flow cytometer to determine transfer between cells of a mobile dye, calcein, as a measure of cell communication through gap junctions. In spite of this being a method with potential for quantitative and reliable determination of GJIC, it has been modestly used, possibly due to technical difficulties. In the present work we have illustrated several ways to use flow cytometric data to express cell communication through gap junctions. The recipient cells were pre-stained with the permanent lipophilic dye PKH26, and the donor cell population were loaded with the gap junction permeable dye, calcein. We show that the method may be used to measure the effect of chemicals on GJIC, and that the information is reliable, objective and reproducible due to the large number of cells studied. The data may give additional information to that obtained with other methods, since the effect observed will be on the establishment of cell communication as compared to what is observed for microinjection or scrape loading, where the effect is on already established communication. This is probably the reason for the more potent effects of DMSO on GJIC measured by the present method than on already existing GJIC measured by microinjection or quantitative scrape loading. We also show that the problem related to the mobile dye calcein not being fixable with aldehydes will not affect the results as long as the cells are kept on ice in the dark and analysed by flow cytometer within the first hours after formalin cell fixation.     

Quantitative determination of the induction of apoptosis in a murine B cell line using flow cytometric bivariate cell cycle analysis.

WEHI-231 cells have been used extensively as a model of tolerance induction in B cells. Recent evidence has shown that anti-IgM treatment of WEHI-231 cells resulted in the induction of apoptosis. In this study, using acridine orange staining and flow cytometric analysis, we demonstrated that apoptotic cells are detected as a distinct population of cells separate from the cells in normal cell cycle progression. The validity of analysis gates was confirmed by cell sorting of the apoptotic population versus normal cells and subsequent gel analysis. Using this technique, we have demonstrated that F(ab')2 anti-mu, A23187, or PMA induced apoptosis in the WEHI-231 cells. The addition of LPS reversed apoptotic induction as seen previously with the WEHI-231 cell line. In contrast, however, PMA did not prevent the induction of apoptosis in anti-mu-treated cells. Additionally, we were interested in determining if the induction of apoptosis was restricted to a specific phase of cell cycle. Since growth inhibition results in most cells arresting in the G1 phase of cell cycle, we wanted to demonstrate apoptosis as a G1-dependent event. This was examined with WEHI-231 cells treated with known cell cycle inhibitors. Interestingly, inhibition of cells in each phase of cycle resulted in the induction of apoptosis. LPS was able to inhibit the induction of apoptosis with each of the cell cycle inhibitors except actinomycin D. Furthermore, we have demonstrated that the WEHI-231 cells contain a Ca(2+)-Mg(2+)-dependent preexisting endonuclease.     

Accurate flow cytometric membrane potential measurement in bacteria using diethyloxacarbocyanine and a ratiometric technique.

BACKGROUND: Membrane potential (MP) plays a critical role in bacterial physiology. Existing methods for MP estimation by flow cytometry are neither accurate nor precise, due in part to the heterogeneity of size of the particles analyzed. The ratio of a size- and MP-sensitive measurement, and an MP-independent, size-sensitive measurement, should provide a better estimate of MP. METHODS: Flow cytometry and spectrofluorometry were used to detect red (488 --> 600 nm) fluorescence associated with aggregates of diethyloxacarbocyanine (DiOC2(3)), which, in the monomeric state, is normally green (488 --> 530 nm) fluorescent. RESULTS: In bacteria incubated with 30 microM dye, aggregate formation increases with the magnitude of the interior-negative membrane potential. Green fluorescence from stained bacteria predominantly reflects particle size, and is relatively independent of MP, whereas red fluorescence is highly dependent on both MP and size. The ratio of red to green fluorescence provides a measure of MP that is largely independent of cell size, with a low coefficient of variation (CV). Calibration with valinomycin and potassium demonstrates that the method is accurate over the range from -50 mV through -120 mV; it also accurately tracks reversible reductions in MP produced by incubation at 4 degrees C and washing in glucose-free medium. CONCLUSIONS: The ratiometric technique for MP estimation using DiOC2(3) is substantially more accurate and precise than those previously available, and may be useful in studies of bacterial physiology and in investigations of the effects of antibiotics and other agents on microorganisms.     

The initiation of mast cell degranulation: activation at the cell membrane.

The low molecular weight mast cell activator, polymyxin B, has been covalently bound to an insoluble matrix of Sepharose 4B. It has been demonstrated that mast cells in preparations of rat peritoneal cells bind to Sepharose 4B-polymyxin B beads but not to control beads. The bound cells are stimulated to degranulate by this interaction at the cell membrane with the resultant release of biogenic amines.     

A flow cytometric assay for intracellular nonprotein thiols using mercury orange

The level of nonprotein thiols was assayed in individual mammalian cells using flow cytometry. Previous determinations of glutathione (GSH, the most abundant nonprotein thiol in most cells) by flow cytometry were based on UV laser excitation of fluorochromes. Because of several shortcomings of UV excitation, an assay for GSH using visible light is of interest. Selective staining of nonprotein thiols with mercury orange (a mercurial compound that binds stoichiometrically to sulfhydryl groups) was obtained by restricting the staining time. By using various drugs that affect GSH levels and overall thiol levels in cells, it was shown that GSH is the primary thiol group being stained. Thus a quick, specific technique using mercury orange has been developed for the flow cytometric determination of nonprotein thiols and preferentially for GSH in individual mammalian cells.     

Refinement of the dichlorofluorescein assay for flow cytometric measurement of reactive oxygen species in irradiated and bystander cell populations

Reactive oxygen species (ROS) have been implicated in many ionizing radiation-related phenomena, including bystander effects. The oxidation of 2'7'-dichlorofluorescin (DCFH) to fluorescent 2'7'-dichlorofluorescein (DCF) is commonly used for the detection of radiation-induced ROS. The DCF assay was adapted for efficient, systematic flow cytometry quantification of low-linear energy transfer (LET) gamma-radiation-induced ROS in vitro in Chinese hamster ovary (CHO) cells. This method is optimized for increased sensitivity to radiation-induced ROS and to discriminate against measurement of extracellular ROS. This method can detect a significant increase in ROS in cells exposed to gamma radiation at doses as low as 10 cGy. The antioxidants N-acetyl-cysteine and ascorbic acid (vitamin C) significantly reduced the amount of ROS measured in cells exposed to 5 Gy ionizing radiation. This method was used to measure the intracellular ROS in unirradiated CHO bystander cells co-cultured with low-LET-irradiated cells. No increase in ROS was measured in bystander cell populations co-cultured with the irradiated cells beginning 9 s after radiation exposure.     

Green fluorescent protein-propidium iodide (GFP-PI) based assay for flow cytometric measurement of bacterial viability.

BACKGROUND: Several staining protocols have been developed for flow cytometric analysis of bacterial viability. One promising method is dual staining with the LIVE/DEAD BacLight bacterial viability kit. In this procedure, cells are treated with two different DNA-binding dyes (SYTO9 and PI), and viability is estimated according to the proportion of bound stain. SYTO9 diffuses through the intact cell membrane and binds cellular DNA, while PI binds DNA of damaged cells only. This dual-staining method allows effective separation between viable and dead cells, which is far more difficult to achieve with single staining. Although SYTO9-PI dual staining is practical for various bacterial viability analyses, the method has a number of disadvantages. Specifically, the passage of SYTO9 through the cell membrane is a slow process, which is significantly accelerated when the integrity of the cell membrane is disrupted. As a result, SYTO9 binding to DNA is considerably enhanced. PI competes for binding sites with SYTO9 and may displace the bound dye. These properties diminish the reliability of the LIVE/DEAD viability kit. In this study, we investigate an alternative method for measuring bacterial viability using a combination of green fluorescent protein (GFP) and PI, with a view to improving data reliability. METHODS: Recombinant Escherichia coli cells with a plasmid containing the gene for jellyfish GFP were stained with PI, and green and red fluorescence were measured by FCM. For comparison, cells containing the plasmid from which gfp was removed were stained with SYTO9 and PI, and analyzed by FCM. Viability was estimated according to the proportion of green and red fluorescence. In addition, bioluminescence and plate counting (other methods to assess viability) were used as reference procedures. RESULTS: SYTO9-PI dual staining of bacterial cells revealed three different cell populations: living, compromised, and dead cells. These cell populations were more distinct when the GFP-PI combination was used instead of dual staining. No differences in sensitivity were observed between the two methods. However, substitution of SYTO9 with GFP accelerated the procedure. Bioluminescence and plate counting results were in agreement with flow cytometric viability data. CONCLUSIONS: In bacterial viability analyses, the GFP-PI combination provided better distinction between current viability stages of E. coli cells than SYTO9-PI dual staining. Additionally, the overall procedure was more rapid. No marked differences in sensitivity were observed.     

Calibration of a flow cytometric assay of glucose-6-phosphate dehydrogenase activity.

The reduction of tetrazolium salts to colored formazans is a reaction which has been exploited both in histo- and cytochemistry. Tetrazolium salts forming fluorescent formazans prove suitable for measuring defined cellular dehydrogenase activities in automated processes. This study considers an important aspect of formazan measurement in flow cytometry, namely, calibration. Calibration is performed by correlating the number (and fluorescence intensity) of formazan-bearing cells measured by flow cytometry with simultaneously performed biochemical analyses of the same material. The method is demonstrated by an example of glucose-6-phosphate dehydrogenase. Using the data of a typical experiment, the enzyme activity is expressed in femtomol of hydrogen transferred per cell during incubation time. Furthermore, through spatially resolved double excitation of formazan and nuclear DAPI fluorescence, an independent analysis of cell cycle and cellular enzymatic activity is established.     

A novel multiparameter flow cytometric assay for inosine triphosphatase expression analysis in leukocytes

The aim of this study was to assess inosine triphosphate (ITPase) expression in the different leukocyte populations present in peripheral blood samples of a nonimmune compromised control group. For this purpose, a multiparameter flow cytometric assay was developed and performed to study ITPase expression in peripheral leukocyte subpopulations of healthy volunteers (n = 20). Qualitative ITPase expression was assessed by determining the percentage of ITPase-positive cells. Quantitative data were obtained by measuring the median fluorescent intensity (MFI). Subcellular localization of ITPase was analyzed using immunocytochemistry. Immunocytochemistry showed that ITPase is present in all leukocytes and localized intracellular. Based on this finding, a multiparameter flow cytometric assay was developed using a Fix & Perm strategy. Qualitative and quantitative ITPase expression remained stable (variation, <10%) for at least 48 h after blood sampling. MFI values showed that activated monocytes contained significantly more ITPase when compared to the total monocyte fraction (P < 0.0001), which subsequently had a higher amount of expression than granulocytes (P < 0.0001). In addition, the phagocyte subpopulations ([activated] monocytes and granulocytes) contained significantly higher levels of ITPase when compared to lymphocytes (P < 0.0001). Within the lymphocyte fraction, it appeared that T-helper cells contained significantly higher ITPase levels when compared to cytotoxic T cells, B lymphocytes, and natural killer cells (P < 0.0001). Our study is the first which describes a flow cytometry assay to analyze ITPase expression in leukocytes qualitatively as well as quantitatively and visualizes the intracellular localization of ITPase in leukocytes. ? 2012 International Society for Advancement of Cytometry.     

The role of mast cell degranulation products in mast cell hyperplasia. I. Mechanism of action of nerve growth factor

A variety of mast cell degranulating agents have previously been shown to induce mast cell hyperplasia in adult rats. In neonates 2.5 S nerve growth factor (NGF) induces a hyperplasia of both mucosal and connective tissue mast cells (MMC and CTMC). We have examined the role of the potent mast cell degranulating properties of NGF on its ability to induce mast cell hyperplasia. Administration of NGF in combination with the mast cell stabilizing agent disodium cromoglycate was found to abrogate the CTMC hyperplasia induced by NGF alone. Treatment of neonatal rats with the alternate degranulating agent compound 48/80 was found to induce a limited CTMC but not a MMC hyperplasia. A supernatant obtained by degranulating purified adult rat peritoneal mast cells with anti-IgE was found to induce hyperplasia of the CTMC population similar to that observed with NGF administration. However, this degranulation product supernatant only induced a limited MMC hyperplasia as judged by RMCP II content of the tissues. These results suggest that NGF has dual action inducing mast cell hyperplasia; CTMC hyperplasia being dependent on the ability of NGF to degranulate mast cells. MMC hyperplasia induced by NGF is independent of CTMC degranulation. Degranulation products from peritoneal mast cells act to increase both MMC and CTMC populations in the neonate. These data suggest that the CTMC population may be regulated by an autocrine positive feedback mechanism in vivo.     

A novel flow cytometric assay to quantify interactions between proteins and membrane lipids

A diverse set of experimental systems has been developed to probe protein-lipid interactions. These include measurements with the headgroups of membrane lipids in solution, immobilized membrane lipids, and analysis of protein binding to membrane lipids reconstituted in liposomes. Each of these methodologies has strengths but also substantial limitations. For example, measurements between proteins and lipid headgroups or with immobilized membrane lipids do not probe interactions in their natural environment, the lipid bilayer. The use of liposomes, however, was so far mostly restricted to biochemical flotation experiments that do not provide quantitative and/or kinetic data. Here, we present a fast and sensitive flow cytometric method to detect protein-lipid interactions. This technique allows for quantitative measurements of interactions between multiple fluorescently labeled proteins and membrane lipids reconstituted in lipid bilayers. The assay can be used to quantify binding efficiencies and to determine kinetic constants. The method is further characterized by a short sampling time of only a few seconds that allows for high-content screening procedures. Finally, using light scatter measurements, the described method also allows for monitoring changes of membrane curvature as well as tethering of liposomes evoked by binding of proteins.     

A novel flow cytometric protocol for assessment of yeast cell adhesion

Microbial adhesion is a field of recognized relevance and, as such, an impressive array of tools has been developed to understand its molecular mechanisms and ultimately for its quantification. Some of the major limitations found within these methodologies concern the incubation time, the small number of cells analyzed, and the operator's subjectivity. To overcome these aspects, we have developed a quantitative method to measure yeast cells' adhesion through flow cytometry. In this methodology, a suspension of yeast cells is mixed with green fluorescent polystyrene microspheres (uncoated or coated with host proteins). Within 2 h, an adhesion profile is obtained based on two parameters: percentage and cells-microsphere population's distribution pattern. This flow cytometry protocol represents a useful tool to quantify yeast adhesion to different substrata in a large scale, providing manifold data in a speedy and informative manner.     

Role of reactive oxygen species in mast cell degranulation

Mast cells are a heterogeneous multifunctional cellular population that promotes connective tissue homeostasis by slow release of biologically active substances, affecting primarily the permeability of vessels and vascular tone, maintenance of electrolyte and water balance, and composition of the extracellular matrix. Along with this, they can rapidly release inflammatory mediators and chemotactic factors that ensure the mobilization of effector innate immune cells to fight against a variety of pathogens. Furthermore, they play a key role in initiation of allergic reactions. Aggregation of high affinity receptors to IgE (FcεRI) results in rapid degranulation and release of inflammatory mediators. It is known that reactive oxygen species (ROS) participate in intracellular signaling and, in particular, stimulate production of several proinflammatory cytokines that regulate the innate immune response. In this review, we focus on known molecular mechanisms of FcεRI-dependent activation of mast cells and discuss the role of ROS in the regulation of this pathway.     

Efficient flow cytometric assay for platelet-leukocyte aggregates in whole blood using fluorescence signal triggering.

BACKGROUND: Platelet-leukocyte aggregates (PLAs) may be important in thrombotic and inflammatory disease states, but accurate assessment of PLA formation in vivo is hampered by the propensity for in vitro artefacts caused by sample manipulation. A whole blood flow cytometric assay for circulating PLAs, based on minimal sample manipulation, was thus developed. METHODS: Citrated whole blood was labeled with a RPE-CD45 MAb (leukocyte marker) and an FITC-CD42a (GPIX) MAb (platelet marker). The latter was used to avoid possible influences of platelet glycoprotein proteolysis by neutrophil-derived proteases. The samples were mildly fixed with 0.5% formaldehyde saline. The cytometer was triggered by RPE-CD45 fluorescence. Leukocyte subpopulations were separated according to their typical light scattering and CD45 expression. RESULTS: Minimal sample manipulation and mild sample fixation resulted in minor in vitro artefacts and good sample stability. Fluorescence triggering increased the efficiency of the flow cytometric analysis approximately 5-fold compared with triggering with light scatter, and allowed discrimination of leukocyte subpopulations. The majority of PLAs involved monocytes and neutrophils, rather than lymphocytes, both without and with in vitro stimulation by ADP or thrombin. A cocktail of blocking MAbs to CD62P, CD15, GPIIb/IIIa and the CD11b/CD18 complex had no effect on unstimulated samples, whilst totally inhibiting aggregation induced by 10(-5) M ADP, suggesting that the PLAs in unstimulated blood were preformed in vivo. CONCLUSIONS: This whole blood flow cytometric assay for PLAs is simple and efficient, and appears to reflect closely platelet-leukocyte aggregates in circulating blood in vivo.