Flow cytometric assay for evaluation of the effects of cell density on cytotoxicity and induction of apoptosis

BACKGROUND: We used a flow cytometric assay, which allows us to perform precise measurements within a wide range of cell concentrations to study the effect of the density of cultured cells on their sensitivity to cytotoxic compounds. METHODS: To measure cytotoxic action, cells are plated in a 96-well plate at a density ranging from 700 to 100,000 cells/ml and are allowed to grow for 72 h in the presence of various concentrations of a cytotoxic agent. To quantitate the number of surviving cells, each sample is analyzed in a flow cytometer with equal acquisition time. Viable cells are identified by light scattering characteristics identical to those for untreated cells. To estimate the amount of viable, apoptotic, or necrotic (late apoptotic) cells, the samples are stained with Annexin V and propidium iodide. RESULTS: Using this method, we found that the cytotoxicity of ascorbic acid for malignant lymphoid CEM-C7 cells can be increased significantly when cell density decreases, reaching a value that is typically lower than the normal physiological concentration of ascorbic acid in blood. CONCLUSION: The flow cytometric analysis described in this study can be useful in comparing the effects of cell density on the cytotoxic action of various compounds.     

Flow cytometric analysis of cell-surface binding elements for fibronectin on mouse lung cell isolates

Fresh lung cell isolates from LAF1 mice were examined for the presence of fibronectin-binding elements using flow cytometric analysis. Thoroughly perfused lungs from adult male mice were dissociated using an elastase-trypsin digestion, gentle pipetting and filtering. The resulting heterogeneous cell suspension was incubated with fibronectin coated 0.5 micron fluorescent beads. Subsequent flow cytometric analysis indicated the presence of two species of specific fibronectin-binding populations; one of higher binding affinity which can be blocked with exogenous plasma fibronectin and one of lower binding affinity. We tentatively identify the lower affinity binding element with the fibronectin adhesion receptor and the higher affinity element with the putative matrix assembly receptor.     

Production of cell lines secreting TAT fusion proteins.

Transduction of proteins and other macromolecules constitutes a potent technology to analyze cell functions and to achieve therapeutic interventions. In general, fusion proteins with protein transduction domains, such as TAT, are produced in a bacterial expression system. Here we describe the generation of a mammalian expression vector coding for TAT-EGFP fusion protein. Transfection of CHO-K1 cells by this vector and subsequent selection by Zeocin resulted in cell lines that express and secrete EGFP, a variant of the green fluorescent protein GFP. The ultimate cell line was produced by first cloning the stable integrants and subsequent selection of EGFP-expressing cells by flow cytometric sorting. In the resulting cell line approximately 98% of cells express EGFP. Using the same methodology, we generated cell lines that express DsRed fluorescent protein. The advantages of using such a mammalian expression system include the ease of generating TAT fusion proteins and the potential for sustained production of such proteins in vitro and, potentially, in vivo.     

Monitoring promoter activity in a single bacterial cell by using green and red fluorescent proteins

We investigated the possibility of monitoring promoter activity with flow cytometry by using green fluorescent protein (GFPmut2) and red fluorescent protein (drFP583) in a single bacterial cell. The drFP583 was used as an intrinsic marker of the bacterial cells, because it was expressed constantly in Escherichia coli MC1061 strain. The GFPmut2 expressed under the control of the Hg(2+) ion inducible mer promoter/operator, was used to study promoter activity. Over 75% of the cells were positive for red and green fluorescence in flow cytometric analysis. The average green fluorescence of the whole population increased from 6.7 to 1700 when the mercury concentration was increased from 0 to 1 x 10(-4) M, while the red fluorescence was unaffected by the mercury concentration. These results show that gfpmut2 and drFP583 could be expressed under different promoters in one bacterial cell and measured independently with a flow cytometer.     

Cellometer image cytometry as a complementary tool to flow cytometry for verifying gated cell populations

Traditionally, many cell-based assays that analyze cell populations and functionalities have been performed using flow cytometry. However, flow cytometers remain relatively expensive and require highly trained operators for routine maintenance and data analysis. Recently, an image cytometry system has been developed by Nexcelom Bioscience (Lawrence, MA, USA) for automated cell concentration and viability measurement using bright-field and fluorescent imaging methods. Image cytometry is analogous to flow cytometry in that gating operations can be performed on the cell population based on size and fluorescent intensity. In addition, the image cytometer is capable of capturing bright-field and fluorescent images, allowing for the measurement of cellular size and fluorescence intensity data. In this study, we labeled a population of cells with an enzymatic vitality stain (calcein-AM) and a cell viability dye (propidium iodide) and compared the data generated by flow and image cytometry. We report that measuring vitality and viability using the image cytometer is as effective as flow cytometric assays and allows for visual confirmation of the sample to exclude cellular debris. Image cytometry offers a direct method for performing fluorescent cell-based assays but also may be used as a complementary tool to flow cytometers for aiding the analysis of more complex samples.     

Flow cytometry in cancer stem cell analysis and separation

In recent years, a special type of cancer cell--the cancer stem cell (CSC)--has been identified and characterized for different tumors. CSCs may be responsible for the recurrence of a tumor following a primarily successful therapy and are thought to bear a high metastatic potential. For the development of efficient treatment strategies, the establishment of reliable methods for the identification and effective isolation of CSCs is imperative. Similar to their stem cell counterparts in bone marrow or small intestine, different cluster of differentiation surface antigens have been characterized, thus enabling researchers to identify them within the tumor bulk and to determine their degree of differentiation. In addition, functional properties characteristic of stem cells can be measured. Side population analysis is based on the stem cell-specific activity of certain ATP-binding cassette transporter proteins, which are able to transport fluorescent dyes out of the cells. Furthermore, the stem cell-specific presence of aldehyde dehydrogenase isoform 1 can be used for CSC labeling. However, the flow cytometric analysis of these CSC functional features requires specific technical adjustments. This review focuses on the principles and strategies of the flow cytometric analysis of CSCs and provides an overview of current protocols as well as technical requirements and pitfalls. A special focus is set on side population analysis and analysis of ALDH activity. Flow cytometry-based sorting principles and future flow cytometric applications for CSC analysis are also discussed.     

Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry

We provide a protocol for a high-resolution flow cytometry-based method for quantitative and qualitative analysis of individual nano-sized vesicles released by cells, as developed and previously described by our group. The method involves (i) bright fluorescent labeling of cell-derived vesicles and (ii) flow cytometric analysis of these vesicles using an optimized configuration of the commercially available BD Influx flow cytometer. The method allows the detection and analysis of fluorescent cell-derived vesicles of ～100 nm. Integrated information can be obtained regarding the light scattering, quantity, buoyant density and surface proteins of these nano-sized vesicles. This method can be applied in nanobiology to study basic aspects of cell-derived vesicles. Potential clinical applications include the detailed analysis of vesicle-based biomarkers in body fluids and quality control analysis of (biological) vesicles used as therapeutic agents. Isolation, fluorescent labeling and purification of vesicles can be done within 24 h. Flow cytometer setup, calibration and subsequent data acquisition can be done within 2-4 h by an experienced flow cytometer operator.     

Plug flow cytometry extends analytical capabilities in cell adhesion and receptor pharmacology

BACKGROUND: Plug flow cytometry is a recently developed system for the automated delivery of multiple small boluses or "plugs" of cells or particles to the flow cytometer for analysis. Important system features are that sample plugs are of precisely defined volume and that the sample vessel need not be pressurized. We describe how these features enable direct cell concentration determinations and novel ways to integrate flow cytometers with other analytical instruments. METHODS: Adhesion assays employed human polymorphonuclear neutrophils (PMNs) loaded with Fura Red and Chinese hamster ovary (CHO) cells cotransfected with genes for green fluorescent protein (GFP) and human P-selectin. U937 cells expressing the human 7-transmembrane formyl peptide receptor were loaded with the fluorescent probe indo-1 for intracellular ionized calcium determinations. A computer-controlled syringe or peristaltic pump loaded the sample into a sample loop of the plug flow coupler, a reciprocating eight-port valve. When the valve position was switched, the plug of sample in the sample loop was transported to the flow cytometer by a pressure-driven fluid line. RESULTS: In stirred mixtures of PMNs and CHO cells, we used plug flow cytometry to directly quantify changes in concentrations of nonadherent singlet PMNs. This approach enabled accurate quantification of adherent PMNs in multicell aggregates. We constructed a novel plug flow interface between the flow cytometer and a cone-plate viscometer to enable real-time flow cytometric analysis of cell-cell adhesion under conditions of uniform shear. The High Throughput Pharmacology System (HTPS) is an instrument used for automated programming of complex pharmacological cell treatment protocols. It was interfaced via the plug flow coupling device to enable rapid (< 5 min) flow cytometric characterization of the intracellular calcium dose-response profile of U937 cells to formyl peptide. CONCLUSIONS: By facilitating the coupling of flow cytometers to other fluidics-based analytical instruments, plug flow cytometry has extended analytical capabilities in cell adhesion and pharmacological characterization of receptor-ligand interactions.     

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

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

Effects of Hoechst 33342 on C2C12 and PC12 cell differentiation

Accumulative evidence demonstrates that normal as well as cancer stem cells can be identified as a side population following Hoechst 33342 staining and flow cytometric analysis. This popular method is based on the ability of stem cells to efflux this fluorescent vital dye. We demonstrate that Hoechst 33342 can affect cell differentiation, suggesting potential complications in the interpretation of data.     

Validation of flow cytometric competitive binding protocols and characterization of fluorescently labeled ligands

BACKGROUND: Fluorescently labeled ligands and flow cytometric methods allow quantification of receptor-ligand binding. Such methods require calibration of the fluorescence of bound ligands. Moreover, binding of unlabeled ligands can be calculated based on their abilities to compete with a labeled ligand. In this study, calibration parameters were determined for six fluorescently labeled N-formyl peptides that bind to receptors on neutrophils. Two of these ligands were then used to develop and validate competitive binding protocols for determining binding constants of unlabeled ligands. METHODS: Spectrofluorometric and flow cytometric methods for converting relative flow cytometric intensities to number of bound ligand/cell were extended to include peptides labeled with fluorescein, Bodipy, and tetramethylrhodamine. The validity of flow cytometric competitive binding protocols was tested using two ligands with different fluorescent properties that allowed determination of rate constants both directly and competitively for one ligand, CHO-NLFNYK-tetramethylrhodamine. RESULTS: Calibration parameters were determined for six fluorescently-labeled N-formyl peptides. Equilibrium dissociation constants for these ligands varied over two orders of magnitude and depended upon the peptide sequence and the molecular structure of the fluorescent tag. Kinetic rate constants for CHO-NLFNYK-tetramethylrhodamine determined directly or in competition with CHO-NLFNYK-fluorescein were statistically identical. CONCLUSIONS: Combination of spectrofluorometric and flow cytometric methods allows convenient calculation of calibration parameters for a series of fluorescent ligands that bind to the same receptor site. Competitive binding protocols have been independently validated.     

Simultaneous flow cytometric analyses of enhanced green and yellow fluorescent proteins and cell surface antigens in doubly transduced immature hematopoietic cell populations

BACKGROUND: Cell transduction with multiple genes offers opportunities to investigate specific gene interactions on cell function. Detection of multiple transduced genes in hematopoietic cells requires strategies to combine measurements of gene expression with phenotypic cell discriminants. We describe simultaneous flow cytometric detection of two green fluorescent protein (GFP) variants in immunophenotypically defined human hematopoietic subpopulations using only a minor physical adjustment to a standard FACSCalibur. METHODS: The accuracy and sensitivity of enhanced GFP (EGFP) and enhanced yellow fluorescent protein (EYFP) detection in mixtures of transduced and nontransduced PG13 packaging cells were evaluated by flow cytometry. Retroviral vectors encoding EGFP or EYFP were used to transduce CD34(+) hematopoietic cells derived from umbilical cord blood. The transduction efficiency into subpopulations of hematopoietic cells was measured using multivariate flow cytometry. RESULTS: A bicistronic retroviral vector containing the EGFP and puromycin N-acetyltransferase (pac) genes afforded brighter EGFP signals in transduced cells than a retroviral vector encoding a pac-EGFP fusion protein. The sensitivity of detecting EGFP and EYFP-expressing cells among a background of nonexpressing cells was 0.01% and 0.05%, respectively. EGFP or EYFP was expressed in up to 95% of CD34(+) DR(-) or CD34(+) 38(-) subpopulations in cord blood 48 h posttransduction. Simultaneous transduction with EGFP and EYFP viral supernatants (1:1 mixture) led to coexpression of both GFP variants in 15% of CD34(+) DR(-) and 20% of CD34(+) 38(-) cells. CONCLUSIONS: These results demonstrate simultaneous detection of EGFP and EYFP in immunophenotypically discriminated human hematopoietic cells. This technique will be useful to quantify transduction of multiple retroviral constructs in discriminated subpopulations.     

DNA flow cytometry in sperm cells from unilaterally orchiectomized patients with testicular cancer before further treatment

Light microscopic sperm cell analysis and DNA flow cytometry in the seminal fluid were done in 85 testicular cancer patients after orchiectomy before further treatment. The results were compared with those from 26 healthy age-matched males (control group). A computer-based method for analysis of the DNA histograms was developed for evaluation of the percentage of sperm cells within the sub-haploid, haploid (1c), and diploid (2c) and greater than 2c levels. Compared with the control group, testicular cancer patients had a reduced sperm cell density and sperm cell motility. The mobility grade was also significantly reduced as compared with healthy males. In addition, the number of condensed haploid sperm cells (within the subhaploid level) was decreased in testicular cancer patients, whereas the percentages of noncondensed haploid (1c), diploid, and greater than 2c cells were increased. Most of the DNA flow cytometric parameters were significantly correlated with sperm cell density. DNA flow cytometry in human seminal fluid offers a possible means of assessing spermatogenesis, thus providing an objective method for studying fertility disturbances, for example, in cancer patients before and after treatment.     

Fluorescence resonance energy transfer analysis of cell surface receptor interactions and signaling using spectral variants of the green fluorescent protein

BACKGROUND: Fluorescence resonance energy transfer (FRET) is a powerful technique for measuring molecular interactions at Angstrom distances. We present a new method for FRET that utilizes the unique spectral properties of variants of the green fluorescent protein (GFP) for large-scale analysis by flow cytometry. METHODS: The proteins of interest are fused in frame separately to the cyan fluorescent protein (CFP) or the yellow fluorescent protein (YFP). FRET between these differentially tagged fusion proteins is analyzed using a dual-laser FACSVantage cytometer. RESULTS: We show that homotypic interactions between individual receptor chains of tumor necrosis factor receptor (TNFR) family members can be detected as FRET from CFP-tagged receptor chains to YFP-tagged receptor chains. Noncovalent molecular complexation can be detected as FRET between fusions of CFP and YFP to either the intracellular or extracellular regions of the receptor chains. The specificity of the assay is demonstrated by the absence of FRET between heterologous receptor pairs that do not biochemically associate with each other. Interaction between a TNFR-like receptor (Fas/CD95/Apo-1) and a downstream cytoplasmic signaling component (FADD) can also be demonstrated by flow cytometric FRET analysis. CONCLUSIONS: The utility of spectral variants of GFP in flow cytometric FRET analysis of membrane receptors is demonstrated. This method of analyzing FRET allows probing of noncovalent molecular interactions that involve both the intracellular and extracellular regions of membrane proteins as well as proteins within the cells. Unlike biochemical methods, FRET allows the quantitative determination of noncovalent molecular associations at Angstrom level in living cells. Moreover, flow cytometry allows quantitative analyses to be carried out on a cell-by-cell basis on large number of cells. Published 2001 Wiley-Liss, Inc.     

Model-based estimation of myeloid hematopoietic progenitor cells in ex vivo cultures for cell and gene therapies

Ex vivo production of hematopoietic progenitor cells has potential applications for cell therapy to alleviate cytopenias associated with chemotherapy and for gene therapy. In both therapies, progenitor and stem cells are considered crucial factors for therapeutic success. Assays for progenitor cells, however, take 2 weeks to complete, which is similar to the length of a typical culture. Therefore, a real-time estimation of the percentage or number of progenitor cells, based on rapid measurements, would be useful for optimization of feeding and harvest decisions. In this study, metabolic activity assays and flow cytometric analysis were used to estimate the content of progenitor cells. The measured metabolic activities are a collective contribution from all types of cells. Cells in granulomonocytic cultures have been lumped into six cell types and metabolic rates have been modeled as a linear function of cell composition and growth rate and as a nonlinear function of cell density. Data from 24 experiments were utilized to determine the model parameters in a calibration step. These data include flow cytometric analysis of more mature hematopoietic cells, progenitor cell colony assays, total cell content, and metabolite concentrations, and cover a wide range of cell composition, cell density, and growth rate. After calibration, the model is able to deliver good predictions of progenitor cell content for cultures with higher percentages of progenitor cells, as well as the peak progenitor cell content, based only on parameters that can be rapidly measured. With the aid of those predictions a harvest strategy was developed that will allow optimizing the harvest time based on the culture kinetics of each patient or donor inoculum, rather than using retrospective analysis to determine a uniform harvest time.     

Fluorescence kinetics in HeLa cells after treatment with cell cycle arrest inducers visualized with Fucci (fluorescent ubiquitination-based cell cycle indicator)

Fucci (fluorescent ubiquitination-based cell cycle indicator) is able to visualize dynamics of cell cycle progression in live cells; G1- and S-/G2-/M-phase cells expressing Fucci emit red and green fluorescence, respectively. This system could be applied to cell kinetic analysis of tumour cells in the field of cancer therapy; however, it is still unclear how fluorescence kinetics change after various treatments, including exposure to anticancer agents. To explore this, we arrested live HeLa cells expressing the Fucci probes at various cell cycle stages and observed the fluorescence, in conjunction with flow cytometric analysis. X-irradiation, HU (hydroxyurea) and nocodazole arrest cells at G2/M boundary, early S-phase and early M-phase, respectively. Although X-irradiation and HU treatment induced similar accumulation kinetics of green fluorescent cells, nocodazole treatment induced an abnormal red fluorescence at M phase, followed by accumulation of both red and green fluorescent cells with 4N DNA content. We conclude that certain agents that disrupt normal cell cycle regulation could cause unexpected fluorescence kinetics in the Fucci system.     

Development of a Robust Flow Cytometric Assay for Determining Numbers of Viable Bacteria

Several fluorescent probes were evaluated as indicators of bacterial viability by flow cytometry. The probes monitor a number of biological factors that are altered during loss of viability. The factors include alterations in membrane permeability, monitored by using fluorogenic substrates and fluorescent intercalating dyes such as propidium iodide, and changes in membrane potential, monitored by using fluorescent cationic and anionic potential-sensitive probes. Of the fluorescent reagents examined, the fluorescent anionic membrane potential probe bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC(inf4)(3)] proved the best candidate for use as a general robust viability marker and is a promising choice for use in high-throughput assays. With this probe, live and dead cells within a population can be identified and counted 10 min after sampling. There was a close correlation between viable counts determined by flow cytometry and by standard CFU assays for samples of untreated cells. The results indicate that flow cytometry is a sensitive analytical technique that can rapidly monitor physiological changes of individual microorganisms as a result of external perturbations. The membrane potential probe DiBAC(inf4)(3) provided a robust flow cytometric indicator for bacterial cell viability.     

Green fluorescent protein (GFP)-dependent separation of bacterial ghosts from intact cells by FACS

BACKGROUND: E. coli and Salmonella ghost preparations, produced by applying the PhiX174 protein E-mediated lysis system, contain nonlysed bacteria at a very low percentage. To use the ghosts as vaccines, additional methods have to be identified to remove any viable cell, to end up in totally inactivated ghost fractions. Materials and Methods To increase the purity of ghost fractions, we established a green fluorescent protein (GFP)-dependent "in vivo staining" method to be combined with the E-mediated lysis system. Several gfp expression vectors were constructed, and the corresponding cellular fluorescence was analyzed. Bacterial fluorescence, exclusively preserved in nonlysed cells, was utilized to separate these cells from ghost preparations via flow cytometric sorting. RESULTS: High-level production of GFP prior to induction of the lysis system did not affect bacterial growth rates and caused no inhibitory effects on the subsequent protein E-mediated lysis of the cells. The population of reproductive or inactivated but nonlysed cells was highly fluorescent at mean intensities 215-fold higher than ghosts, which exhibited fluorescence at background level. Fluorescent cells could effectively be separated from ghost preparations via flow cytometric sorting. Cell sorting subsequent to protein E-mediated lysis reduced the number of viable cells within ghost preparations by a factor of 3 x 10(5). CONCLUSIONS: The presented procedure is compatible with the protein E-mediated lysis system, is highly effective in separation of nonlysed fluorescent cells, and may serve as a prototype for ghost-purification in applications where only a minimum number of viable cells within ghost preparations can be tolerated.     

X-ray- and neutron-induced chromosome damage detected by flow cytometry compared to cell lethality and chromosome structural changes

V79 Chinese hamster cells were irradiated in G0 phase with 200 kV X rays or 14 MeV neutrons, and dose-response curves were determined for three end points: chromosome damage detected by flow cytometric analysis of chromosomes isolated from metaphase cells in irradiated cultures; loss of clonogenic capacity; and induction of dicentric, tricentric, and ring chromosomes. The changes observed in the flow karyotypes from irradiated cultures were quantitatively evaluated by computer analysis. Estimates of the frequencies of chromosome lesions were derived from an analysis of the flow cytometric measurements by means of a comparison with model calculations simulating the effect of chromosome changes on flow karyotypes. The results indicate that lesions assayed by flow cytometry occur three times more frequently than lethal lesions, while the chromosomal structural changes detected by microscopic analysis were about 10 times less frequent than the lesions detected by flow cytometry. Dose-response curves for X rays and neutrons show that cell reproductive death and changes in flow karyotypes result from damage, induced with a similar relative biological effectiveness. Dose-effect relations derived from changes in flow karyotypes, which can be obtained within 24 h after irradiation, might be of value as a predictive test for the sensitivity of cells for loss of clonogenic capacity.     

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.