Laser flow cytometry and cancer chemotherapy: detection of intracellular anthracyclines by flow cytometry.

The intracellular distribution of important chemotherapeutic antibiotics belonging to the anthracycline group (e.g. adriamycin) can be detected by laser flow cytometry. The indirect method is based on the interference of these compounds with the binding of propidium iodide to the nuclear DNA. While in the direct method, the intracellular fluorescence of these antibiotics is excited and detected with a laser beam in a flow system. The present report demonstrates the use of these two methods for intracellular detection and quantitation of a number of important anthracyclines.     

Analysis of UV-excited fluorochromes by flow cytometry using near-ultraviolet laser diodes.

INTRODUCTION: Violet laser diodes have become common and reliable laser sources for benchtop flow cytometers. While these lasers are very useful for a variety of violet and some ultraviolet-excited fluorochromes (e.g., DAPI), they do not efficiently excite most UV-stimulated probes. In this study, the next generation of InGaN near-UV laser diodes (NUVLDs) emitting in the 370-375-nm range have been evaluated as laser sources for cuvette-based flow cytometers. METHODS: Several NUVLDs, ranging in wavelength from 370 to 374 nm and in power level from 1.5 to 10 mW, were mounted on a BD Biosciences LSR II and evaluated for their ability to excite cells labeled with the UV DNA binding dye DAPI, several UV phenotyping fluorochromes (including Alexa Fluor 350, Marina Blue, and quantum dots), and the fluorescent calcium chelator indo-1. RESULTS: NUVLDs at the 8-10-mW power range gave detection sensitivity levels comparable to more powerful solid-state and ion laser sources, using low-fluorescence microsphere beads as measurement standards. NUVLDs at all tested power levels allowed extremely high-resolution DAPI cell cycle analysis, and sources in the 8-10-mW power range excited Alexa Fluor 350, Marina Blue, and a variety of quantum dots at virtually the same signal-to-noise ratios as more powerful UV sources. CONCLUSIONS: These evaluations indicate that near-UV laser diodes installed on a cuvette-based flow cytometer performed nearly as well as more powerful solid-state UV lasers on the same instrumentation, and comparably to more powerful ion lasers on a jet-in-air system, and. Despite their limited power, integration of these small and inexpensive lasers into benchtop flow cytometers should allow the use of flow cytometric applications requiring UV excitation on a wide variety of instruments.     

Surface labelling of plant protoplasts with fluorochromes for discrimination of heterokaryons by microscopy and flow cytometry

Surface labelling of plant protoplasts was tested for use in mass fusion systems and heterokaryon detection. Parameters have been established for biotinylation and subsequent incubation with avidin-coupled fluorochromes. The procedure is rapid (less than 3 hours) and does not affect viability. Fusion responses were the same as with unlabelled protoplasts. From a range of fluorochromes tested, fluorescein and phycoerythrin proved best suited for detection experiments with protoplasts of both suspension and leaf origin. With this standard combination of labels, as applied in experiments with animal cells, heterokaryons from fused plant protoplasts could clearly be discriminated from other protoplasts by means of fluorescence microscopy or flow cytometry with a single combination of filters and wavelengths.     

Improved methodology for intracellular enzyme reaction and inhibition kinetics by flow cytometry

Flow cytoenzymology is the determination of enzyme activities or concentrations in single intact cells. Using the flow cytometer built and designed in our laboratory and recent modifications to hardware and software, we have developed an improved dynamic flow cytoenzymological procedure for the assay of cellular enzyme kinetics. The reaction mixture is sampled continuously, and the computer clock incorporates time as a parameter for kinetic determinations. Conditions for cellular esterase analysis were optimized and the rates of hydrolysis of two fluorogenic substrates, fluorescein diacetate (FDA) and 4-methylumbelliferone acetate (MUA), by esterases in EMT6 mouse mammary tumor cells were studied. Reaction kinetics were characterized, and Km values of 19 and 72 microM were obtained for the hydrolysis of FDA and MUA respectively. The kinetics of the cellular efflux of fluorescein were investigated, and a half-life of 7.5 min obtained. Enzyme inhibition kinetics were investigated using the competitive substrates p-nitrophenyl acetate and phenyl acetate, and the carbamoylating agents physostigmine and n-butyl isocyanate. The latter was particularly potent with an I50 of 4.8 X 10(-5) M for FDA hydrolysis compared with 6.5 X 10(-3) M for physostigmine. The I50 of 8.8 X 10(-5) M for n-butyl isocyanate inhibition of MUA hydrolysis was similar to that obtained with FDA as substrate. By monitoring FDA and MUA reactions separately and simultaneously, we showed them to act as competitive substrates. A comparison of flow cytoenzymological and conventional spectrofluorimetric analysis was also made, and differences identified in some cases.     

Direct estimation of serological H-Y antigen by flow cytometry

Summary This study was undertaken to evaluate the efficiency of flow cytometry in the detection of the serological H-Y antigen, and to survey expression of HY in the normal human population. Peripheral blood leukocytes (granulocytes) were reacted with monoclonal H-Y antibody, gw-16, and with FITC-conjugated goat anti-mouse IgG, and then were scored for fluorescence in the flow cytometer. Assigned H-Y phenotype was correlated accurately with sex phenotype in 33 out of 38 women and 62 out of 65 men. Data were evaluated by exploratory data analysis (EDA).     

Application of flow cytometry to algal physiology and phytoplankton ecology

Abstract Flow cytometry, originally developed for biomedical purposes, is now being put to extensive use in the study of microalgae. Successful applications to date include studies of cytochemistry, cell cycle dynamics and phytoplankton ecology. This review summarises recent work in a rapidly expanding field of research, and highlights those areas in which significant progress can be expected when the necessary equipment becomes more readily available.     

Particle dispersion for further Cryptosporidium and Giardia detection by flow cytometry

AIMS: The aim of this study was to overcome the analytical problems encountered during the detection of protozoans by flow cytometry resulting from particle compaction. METHODS AND RESULTS: Malvern Mastersizer (Malvern Instruments, Malvern, UK) was used to characterize the particle distribution of four different water samples and/or particle concentrates incubated with (i) low ionic strength solution or sequestring agent, (ii) anionic or non-ionic surfactants (iii) industry detergent formulations and (iv) physical treatment. The recovery of oocysts and cysts in seeded and treated particle concentrates was estimated by cytometry and microscopy. The decrease in ionic strength of the aqueous solution was most efficient in particle dispersion for different types of water. Moreover, samples treated with deionized water or tetrasodium pyrophosphate showed the highest recovery with more than 80% of the oocysts and cysts recovered. CONCLUSIONS: Chemical treatments that act by altering the ionic strength of the medium are the most efficient for all water types tested here but the overall detergency performance cannot be predicted for all water types. SIGNIFICANCE AND IMPACT OF THE STUDY: Flow cytometric detection has been replaced largely by immunomagnetic separation but the data recorded still have relevance in this technique as well as in molecular techniques requiring DNA or RNA extraction.     

Platelet activation as a marker for in vivo prothrombotic activity: detection by flow cytometry

Circulating platelets play a pivotal role in hemostasis. The platelet hemostatic function involves the direct interaction with damaged vessel walls, and circulating coagulation factors, primarily thrombin resulting in platelet activation, aggregation and formation of hemostatic plug. Flow cytometry is a useful technique for the study of platelet activation in circulating blood. Platelet activation markers for ex vivo analysis may include a) activation-dependent epitopes of the membrane glycoprotein (GP) IIb/IIIa (CD41a) receptor, as demonstrated by the binding of activation-specific monoclonal antibodies (MoAbs) PAC1, anti-LIBS1 and anti-RIBS); b) the expression of P-selectin (CD62p), the alpha-granule GP translocated to the platelet surface following release reaction; and c) platelet procoagulant activity, as demonstrated by the binding of i) annexin V protein to the prothrombinase-complex (prothrombin, activated factor X (Xa) and V (Va)) binding sites on the surface of activated platelets, and of ii) MoAbs against activated coagulation factors V and X bound to the surface of activated platelets. Using this method, platelet activation as a marker for in vivo prothrombotic activity can be demonstrated in various clinical conditions including coronary angioplasty, orthostatic challenge in primary depression, sickle cell disease in clinical remission and during pain episode, and in pregnancy-related hypertension with marked increase during preeclampsia. The finding of platelet procoagulant activity is corroborated by increased levels of plasma markers for thrombin generation and fibrinolytic activity.     

流式细胞技术在微生物检测领域的应用研究

研究应用流式细胞技术(flow cytometry method,FCM)进行快速微生物检测的方法。与传统微生物检测方法相比,FCM法更快速和准确。经Pearson相关系数分析表明,在一定浓度范围内,FCM检测法与标准平板检测法(SPC)具有极强线性相关性。经Q检验法分析,FCM检测法具有良好的重复性。由此可见,FCM法可成为一种替代传统微生物检测法的自动化仪器检测新技术。     

Effect of cyclosporin A and ionophores on the intracellular pH of lymphocytes as measured by flow cytometry

Because of the known importance of pHi changes on the biology of cells, we have investigated the effect of CsA, the clinically important immunomodulator, on the pHi of resting mouse spleen and human peripheral blood lymphocytes. We have found that pharmacological doses (0.8 microM) of CsA caused no change in the pHi in these cells. Similarly, the Ca2+ ionophore, A23187, up to 5 microns concentration, had no effect on the pHi of these cells, in contrast to previous reports. However, higher doses of CsA (2.4-8.0 microM) did change the pHi transiently or permanently. These results were obtained with flow cytometric measurements of fluorescence intensity of the pH-sensitive dye, BCECF.     

The binding kinetics and interaction of DNA fluorochromes used in the analysis of nuclei and chromosomes by flow cytometry

The interactions and binding characteristics of DNA dyes used in the flow cytometric analysis of chromatin were studied using human chromosomes and mouse thymocyte nuclei. The kinetics of dye binding and the relationship between fluorescence intensity and dye concentration are presented. Under the conditions used, Hoechst 33258, propidium iodide and chromomycin A3 reach an equilibrium with thymocyte nuclei after approximately 5 min, 20 min and more than 1 h, respectively. The same binding kinetics are observed with Hoechst 33258 and chromomycin when nuclei are stained with a mixture of the two dyes. Sodium citrate, which improves the resolution of flow karyotypes, causes a rapid increase in Hoechst and propidium iodide fluorescence, but a decrease in the fluorescence of chromomycin. The relative peak positions of chromosomes in a flow karyotype are unaffected by sodium citrate addition. The spectral interaction between Hoechst and chromomycin is quantified. There is variation among the human chromosome types in the amount of energy transferred from Hoechst to chromomycin. By measuring the Hoechst and chromomycin fluorescence of each chromosome after Hoechst excitation, it is shown that the amount of energy transferred is correlated to the ratio of the amount of Hoechst to chromomycin bound. Although the energy transfer between the two dyes is considerable, this has little effect on the reproducibility of flow karyotype measurements. The relative peak positions of all human chromosomes in a 64 X 64 channel flow karyotype, except for the 13 and Y chromosomes, vary in the order of 0.5 channel over a 16-fold change in either Hoechst or chromomycin concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

The binding kinetics and interaction of DNA fluorochromes used in the analysis of nuclei and chromosomes by flow cytometry

Summary The interactions and binding characteristics of DNA dyes used in the flow cytometric analysis of chromatin were studied using human chromosomes and mouse thymocyte nuclei. The kinetics of dye binding and the relationship between fluorescence intensity and dye concentration are presented. Under the conditions used, Hoechst 33258, propidium iodide and chromomycin A3 reach an equilibrium with thymocyte nuclei after approximately 5 min, 20 min and more than 1 h, respectively. The same binding kinetics are observed with Hoechst 33258 and chromomycin when nuclei are stained with a mixture of the two dyes. Sodium citrate, which improves the resolution of flow karyotypes, causes a rapid increase in Hoechst and propidium iodide fluorescence, but a decrease in the fluorescence of chromomycin. The relative peak positions of chromosomes in a flow karyotype are unaffected by sodium citrate addition. The spectral interaction between Hoechst and chromomycin is quantified. There is variation among the human chromosome types in the amount of energy transferred from Hoechst to chromomycin. By measuring the Hoechst and chromomycin fluorescence of each chromosome after Hoechst excitation, it is shown that the amount of energy transferred is correlated to the ratio of the amount of Hoechst to chromomycin bound. Although the energy transfer between the two dyes is considerable, this has little effect on the reproducibility of flow karyotype measurements. The relative peak positions of all human chromosomes in a 64×64 channel flow karyotype, except for the 13 and Y chromosomes, vary in the order of 0.5 channel over a 16-fold change in either Hoechst or chromomycin concentration. This implies that, with the present flow cytometers, variation in staining conditions will have minimal effects on the reproducibility of the relative peak positions in flow karyotypes.In honour of Prof. P. van Duijn     

Analysis of free intracellular calcium by flow cytometry: multiparameter and pharmacologic applications

Flow cytometry offers numerous advantages over traditional techniques for measuring intracellular Ca(2+) in lymphoid and nonlymphoid cells. In particular, the heterogeneity of cell responses can be defined by flow cytometry, and multiparameter analyses permit the determination of intracellular Ca(2+) in surface-marker-defined target cells as well as correlation of changes in Ca(2+) with other biochemical markers, including ligand binding. This article presents several established methods for measuring intracellular Ca(2+) by flow cytometry in lymphoid and nonlymphoid cells. Examples are provided for determination of Ca(2+) in human peripheral blood leukocytes and two human epithelial cell lines grown in monolayer. In addition, applications are reviewed or presented for correlating changes in intracellular Ca(2+) with other cell parameters, including cell cycle analysis, changes in cell membrane integrity, and the induction of apoptosis markers. Finally, a number of novel sample handling capabilities useful for performing kinetic analyses of Ca(2+) changes by flow cytometry are now available and one application is presented which is finding utility in pharmacologic studies.     

Growth kinetics of algal populations exsymbiotic from Paramecium bursaria by flow cytometry measurements.

BACKGROUND: The ciliate Paramecium bursaria normally exists as a green paramecium system because each animal cell carries several hundred, unicellular, green, algal cells in its cytoplasm. One of the remarkable and poorly understood pecularities of this system is the steady state in the number of algae per protozoan cell. A major point in the study of mechanisms governing the persistence of symbiont numbers is adequate understanding of the algal life cycle. METHODS: Asynchronously growing cell populations of several algal strains (SA-1, SA-3, and SA-9) exsymbiotic from P. bursaria were characterized by flow cytometry. Algal endogenous chlorophyll and DNA contents were monitored to analyze cell growth kinetics at logarithmic and stationary culture phases. Cell sorting visualized the morphology of algae corresponding to the hyperhaploid (2C and 4C) DNA peaks. RESULTS: Cell-division cycle-dependent changes in chlorophyll and DNA content distributions were most dramatic in logarithmically growing algal populations (an increase in the number of S-phase cells and cells with more chlorophyll), which are thought to be associated with accelerated DNA and chlorophyll metabolism in log-phase algal cultures. Upon reaching the stationary phase of growth, algal populations distinctly showed, in addition to one haploid (1C) DNA peak, two hyperhaploid peaks (2C and 4C) corresponding mainly to cells with two and four nuclei, respectively. CONCLUSIONS: Growth characteristics of algae exsymbiotic from P. bursaria monitored by flow cytometry provide valuable information for the analysis of the algal life cycle, which is important for understanding the regulation mechanisms of symbiont numbers.     

Rapid titration of retroviral vectors encoding intracellular antigens by flow cytometry.

Flow cytometry was used to detect cells infected with retroviral vectors encoding both simian virus 40 large T antigen and G418 resistance after indirect immunofluorescence staining using a T-antigen-specific monoclonal antibody and a fluorescein-conjugated secondary antibody. Titers of viral stocks determined by flow cytometry were equivalent to those determined by quantitation of G418-resistant colonies.     

Detection of the intracellular ras p21 oncogene product by flow cytometry

Rapid identification of the expression of oncogene products in specific cell types could potentially be useful in the diagnosis and treatment of human malignancy. We have now observed that through the use of lysolecithin permeabilization and fluorescence-activated flow cytometry, cells expressing high levels of the v-Ha-ras oncogene product, p21, can readily be distinguished from the nontransformed parent cells in a rapid and quantitative manner.     

流式细胞术检测胞内重组耻垢分枝杆菌的活力研究

目的建立一种快速检测胞内分枝杆菌活力的方法。方法将一定量培养至对数生长期的含pMV-eis的重组耻垢分枝杆菌感染U937巨噬细胞,以含空质粒的耻垢分枝杆菌为对照,吞噬作用2 h后洗去胞外细菌,再分别培养4、12、24和48 h后收集细胞并裂解之。获得的胞内细菌用FDA荧光染料染色后用流式细胞仪检测死亡率,并与平板菌落计数法进行比较。结果流式细胞仪检测出感染12 h后重组耻垢分枝杆菌胞内死亡率较对照组均有显著下降（P〈0.05）,流式细胞仪检测法与平板菌落计数法相比差异无统计学意义（P〉0.05）。结论流式细胞术与传统的平板计数法相比具有快速、敏感、方便的特点,可用于分枝杆菌活菌快速检测。     

Different nucleolar antigen expression in resting and proliferating human lymphocytes as studied by fluorescence microscopy and flow cytometry

Abstract. The aim of this study was to investigate the use of a nucleolar antigen to discriminate between proliferating and resting cells. Antinucleolar antibodies (Si87) were obtained from a scleroderma patient. The specificity of immunostaining was verified and morphological changes in nucleoli were monitored using a fluorescence microscope. Fluorescence of propidium iodide-stained DNA and nucleolar immunofluorescence were measured by flow cytometry.
Following phytohaemagglutinin stimulation the number of nucleoli of normal human peripheral blood lymphocytes increased about 3-fold, accompanied by enlargement of nucleolar size. Simultaneously a mean increase in total immunofluorescence per cell by a factor of three was detected. The method developed and applied here allows a discrimination between resting and proliferating human lumphocytes on the basis of their nucleolar antigen content.     

Different nucleolar antigen expression in resting and proliferating human lymphocytes as studied by fluorescence microscopy and flow cytometry

The aim of this study was to investigate the use of a nucleolar antigen to discriminate between proliferating and resting cells. Antinucleolar antibodies (Si87) were obtained from a scleroderma patient. The specificity of immunostaining was verified and morphological changes in nucleoli were monitored using a fluorescence microscope. Fluorescence of propidium iodide-stained DNA and nucleolar immunofluorescence were measured by flow cytometry. Following phytohaemagglutinin stimulation the number of nucleoli of normal human peripheral blood lymphocytes increased about 3-fold, accompanied by enlargement of nucleolar size. Simultaneously a mean increase in total immunofluorescence per cell by a factor of three was detected. The method developed and applied here allows a discrimination between resting and proliferating human lymphocytes on the basis of their nucleolar antigen content.