Characterization of antibody binding to three cancer-related antigens using flow cytometry and cell tracking velocimetry

Proper antibody labeling is a fundamental step in the positive selection/isolation of rare cancer cells using immunomagnetic cell separation technology. Using either a two-step or single-step labeling protocol, we examined a combination of six different antibodies specific for three different antigens (epithelial specific antigen, epithelial membrane antigen, and HER-2/Neu) on two different breast cancer cell lines (HCC1954 and MCF-7). When a two-step labeling protocol was used (i.e., anti-surface marker-fluoroscein-isothiocyanate [FITC] [primary Ab], anti-FITC magnetic colloid [secondary Ab]) saturation of the primary antibody was determined using fluorescence intensity measurements from flow cytometry (FCM). The saturation of the secondary antibody (or saturation of a single-step labeling) was determined using magnetophoretic mobility measurements from cell tracking velocimetry (CTV). When the maximum magnetophoretic mobility was the primary objective, our results demonstrate that the quantities necessary for antibody saturation with respect to fluorescence intensity were generally higher than those recommended by the manufacturer. The results demonstrate that magnetophoretic mobility varies depending on the types of cell lines, primary antibodies, and concentration of secondary magnetic colloid-conjugated antibody. It is concluded that saturation studies are a vital preparatory step in any separation method involving antibody labeling, especially those that require the specificity of rare cell detection.     

Fluorescence spectroscopy for detection of malignancy: H-ras overexpressing fibroblasts as a model.

Autofluorescence from intracellular chromophores upon illumination of cells by monochromatic light has been studied towards the development of novel noninvasive and sensitive technology for the early detection of cancer. To investigate the relationship between biochemical and morphological changes underlying malignant disease and resulting fluorescence spectra, an in vitro model system of a paired normal and malignant murine fibroblasts cell lines, differing in cancer-associated H-ras expression was employed. A comparison of fluorescence excitation and emission spectra of proliferative cells revealed that fluorescence intensity of malignant cells was significantly less than that of normal cells upon excitation at 290 nm. Fluorescence of both cell lines decreased with decreasing cell concentration, but at each concentration, normal cells had higher fluorescence intensity than malignant cells. Similar differences between the cell lines were observed when brought to quiescence or at stationary phase. Results suggested that the chromophore contributing most significantly to these spectra is tryptophan and its moieties in proteins. This model system demonstrates the specific contribution of H-ras to subcellular chromophores, resulting in a significant difference in their autofluorescence intensity, and implies the potential use of the technique for cancer detection. This model system is potent for analysis of the contribution of other oncogenes and their combinations towards spectral detection of cancer.     

Extremely low frequency (ELF) pulsed-gradient magnetic fields inhibit malignant tumour growth at different biological levels

Extremely low frequency (ELF) pulsed-gradient magnetic field (with the maximum intensity of 0.6-2.0 T, gradient of 10-100 T.M(-1), pulse width of 20-200 ms and frequency of 0.16-1.34 Hz treatment of mice can inhibit murine malignant tumour growth, as seen from analyses at different hierarchical levels, from organism, organ, to tissue, and down to cell and macromolecules. Such magnetic fields induce apoptosis of cancer cells, and arrest neoangiogenesis, preventing a supply developing to the tumour. The growth of sarcomas might be amenable to such new method of treatment.     

Experimental studies on extremely low frequency pulsed magnetic field inhibiting sarcoma and enhancing cellular immune functions

The previous observation with an electron microscope showed that extremely low frequency (ELF) pulsed magnetic field (PMF) (with the maximum intensity of 0. 6-2. 0 T, gradient of 10-100 T. M-1, pulse width of 20-200 ms and frequency of 0. 16-1. 34 Hz) inhibited the growth of S-180 sarcoma in mice and enhanced the ability of immune cell's dissolving sarcoma cells. In this study, the DNA contents of nuclei were assayed by using Faulgen Staining method. With an electron microscope and cell stereoscopy technology it was observed that magnetic field affected the sarcoma cell's metabolism, lowered its malignancy, and restrained its rapid and heteromorphic growth. The magnetic field enhanced the cellular immune ability and the reaction of lymphocytes and plasma. Since ELF pulsed magnetic fields can inhibit the growth of sarcomas and enhance the cellular immune ability, it is possible to use it as a new method to treat cancer.     

Discrepancies in ploidy determination due to specimen sampling errors

Two techniques are described to enhance the detection of low frequency aneuploid cells in automated cell analysis. One method concerns a cell preparation technique; the other is focused on specific cell selection at the measurement level. The cell preparation method has been designed to select and process the tumour areas in paraffin blocks and can be used for image as well as for flow cytometry. The technique uses incident fluorescence microscopy for visual inspection of the surface of the fluorescently stained tissue block to select the specific tumour parts. Using image cytometry, it is shown that in tissue sections with very small tumour foci and many normal cells, aneuploidy could only be detected after enrichment of the cell sample with the specifically selected areas. The cell selection at the measurement level is directed towards detection of low frequency aneuploid cells on microscope slides using the specific capacities of LEYTAS (Leyden Television Analysis System). With this system, cells of interest can be selected by means of minimum size and intensity thresholds. In addition to measurement of the total cell population, all cells above a minimum DNA value can thus be specifically selected and measured. The advantage of both enrichment techniques is the possibility to detect and measure aneuploid cell lines in cases where normal, diploid cells dominate the paraffin tissue.     

Enumeration and viability of rare cells in a microfluidic disk via positive selection approach

Recent studies have shown that specific rare cells in the blood can serve as an indicator of cancer prognosis, among other purposes. This article demonstrates the concept of separating and detecting rare cells from peripheral blood mononuclear cells via an economical microfluidic disk with a model system. MCF7, labeled with magnetic beads, was used to simulate circulating tumor cells as a target. Jurkat clone E6-1 was used to simulate leukocytes or other cells abundant in human blood. A tailored multistage magnet maximized the magnetic field to ensure optimal trapping efficiency. Results indicate that the yield of detected MCF7 was consistent at approximately 80% when fewer than hundreds of MCF7 cells were mixed in greater than 1 million Jurkat cells. The 80% yield also held for 10 MCF7 in 100million Jurkat (rarity of 10(7)). Compared with the results from autoMACS, the performance was at least 20% higher and was more independent of the number of Jurkat. The viability of the enriched cells was approximately 90±20%, showing that this method caused little damage to trapped cells. The microfluidic disk should be applicable for separation and detection of various rare cells, such as circulating tumor cells and circulating endothelial cells in human blood.     

Effect of deproteinization on the in situ chromatin staining with 7-aminoactinomycin D

The possibility of use of 7-amino-actinomycin D (7aAMD)--fluorescent analog of actinomycin D--as a specific dye for DNA staining in the suspended cells was studied by means of laser flow-cytometry. The optimal conditions for staining were obtained: 7aAMD concentration 10(-5) M, pH 7, staining time 20 min, 37 degrees C, ionic strength 0.15 M Na+. In this case the fluorescent signal is proportional to the DNA amount and coefficient of variation is about 0.03. The influence of the stepwise extraction of the proteins from chromatin also was studied. In the course of the salt deproteinization the fluorescence intensity gradually rose thus showing the increase of the binding sides-number. The deproteinization of cells nuclei by 0.1 HCl increased the number of binding sites 2.5 times more. It was shown that the incubation of cells with RNAse at elevated ionic strength (0.3-0.7 M NaCl) leads to an additional increase of the cell fluorescence and produces no effect at low and normal ionic strength. The deproteinizing effect of RNAse and its possible mechanism is discussed.     

NADH荧光法快速检测细菌总数

基于细菌胞内NADH的荧光特性及其在胞内含量稳定的特性, 建立一种快速检测细菌总数的新方法。该荧光法的NADH检测限为1 nmol/L, NADH含量在10 nmol/L~0.2 mmol/L间与荧光强度呈良好线性关系(R2 =0.9905)。经离心获得菌体细胞, 热Tris-HCl法提取胞内NADH, 以 342 nm为激发波长, 461 nm为发射波长测定提取液荧光强度, 1 h内可检测到样品1×104 CFU/mL菌数。结果表明该方法快速、灵敏、简便、重复性好, 可适用于食品卫生与安全、环境检测等领域活细菌数量的定量检测。     

Detection of E. coli O157:H7 by immunomagnetic separation coupled with fluorescence immunoassay

Conventional culture-based methods for detection of E. coli O157:H7 in foods and water sources are time-consuming, and results can be ambiguous, requiring further confirmation by biochemical testing and PCR. A rapid immunoassay prior to cultivation to identify presumptive positive sample would save considerable time and resources. Immunomagnetic separation (IMS) techniques are routinely used for isolation of E. coli O157:H7 from enriched food and water samples, typically in conjunction with cultural detection followed by biochemical and serological confirmation. In this study, we developed a new method that combines IMS with fluorescence immunoassay, termed immunomagnetic fluorescence assay (IMFA), for the detection of E. coli O157:H7. E. coli O157:H7 cells were first captured by anti-O157 antibody-coated magnetic beads and then recognized by a fluorescent detector antibody, forming an immunosandwich complex. This complex was subsequently dissociated for measurement of fluorescence intensity with Signalyte™-II spectrofluorometer. Experiments were conducted to evaluate both linearity and sensitivity of the assay. Capture efficiencies were greater than 98%, as determined by cultural plating and quantitative real-time PCR, when cell concentrations were <10⁵ cells/mL. Capture efficiency decreased at higher cell concentrations, due to the limitation of bead binding capacity. At lower cell concentrations (10–10⁴ cells/mL), the fluorescence intensity of dissociated Cy5 solution was highly correlated with E. coli 157:H7 cell concentrations. The detection limit was 10 CFU per mL of water. The assay can be completed in less than 3 h since enrichment is not required, as compared to existing techniques that typically require a 24 h incubation for pre-enrichment, followed by confirmatory tests.     

A method for analysing phosphatase activity in aquatic bacteria at the single cell level using flow cytometry

It has been demonstrated that ELF97-phosphate (ELF-P) is a useful tool to detect and quantify phosphatase activity of phytoplankton populations at a single cell level. Recently, it has been successfully applied to marine heterotrophic bacteria in culture samples, the cells exhibiting phosphatase activity being detected using epifluorescence microscopy. Here, we describe a new protocol that enables the detection of ELF alcohol (ELFA), the product of ELF-P hydrolysis, allowing the detection of phosphatase positive bacteria, using flow cytometry. Bacteria from natural samples must be disaggregated and, in oligotrophic waters, concentrated before they can be analyzed by flow cytometry. The best efficiency for disaggregating/separating bacterial cell clumps was obtained by incubating the sample for 30 min with Tween 80 (10 mg l(-1), final concentration). A centrifugation step (20,000 g; 30 min) was required in order to recover all the cells in the pellet (only 7+/-2% of the cells were recovered from the supernatant). The cells and the ELFA precipitates were resistant to these treatments. ELFA-labelled samples were stored in liquid nitrogen for up to four months before counting without any significant loss in total or ELFA-labelled bacterial cell abundance or in the ELFA fluorescence intensity. We describe a new flow cytometry protocol for detecting and discriminating the signals from both ELFA and different counterstains (4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI)) necessary to distinguish between ELFA-labelled and non ELFA-labelled heterotrophic bacteria. The method has been successfully applied in both freshwater and marine samples. This method promises to improve our understanding of the physiological response of heterotrophic bacteria to P limitation.     

In-situ monitoring of breast cancer cell (MCF-7) growth and quantification of the cytotoxicity of anticancer drugs fluorouracil and cisplatin

A wireless sensing device was developed for the in-situ monitoring of the growth of human breast cancer cells (MCF-7) and evaluation of the cytotoxicity of the anticancer drugs fluorouracil and cisplatin. The sensor is fabricated by coating a magnetoelastic ribbon-like sensor with a layer of polyurethane that protects the iron-rich sensor from oxidation and provides a cell-compatible surface. In response to a time-varying magnetic field, the magnetoelastic sensor longitudinally vibrates, emitting magnetic flux that can be remotely detected by a pick-up coil. No physical connections between the sensor and the detection system are required. The wireless property facilitates aseptic biological operation, especially in cell culture as illustrated in this work. The adhesion of cells on the sensor surface results in a decrease in the resonance amplitude, which is proportional to the cell concentration. A linear response was obtained in cell concentrations of 5x10(4) to 1x10(6)cellsml(-1), with a detection limit of 1.2x10(4)cellsml(-1). The adhesion strength of cells on the sensor is qualitatively evaluated by increasing the amplitude of the magnetic excitation field. And the cytotoxicity of the anticancer drugs fluorouracil and cisplatin is evaluated by the magnetoelastic biosensor. The cytostatic curve is related with the quantity of cytostatic drug. The lethal concentration (LC50) for cells incubated in the presence of drugs for 20h is calculated to be 19.9muM for fluorouracil and 13.1muM for cisplatin.     

Detection of rare MCF-7 breast carcinoma cells from mixtures of human peripheral leukocytes by magnetic deposition analysis.

BACKGROUND: The presence of malignant breast cancer cells in bone marrow or peripheral blood is a prognostic factor. We tested the capacity of a novel magnetic cell analyzer to detect rare cancer cells in mixtures with human peripheral leukocytes. METHODS: Human peripheral leukocytes were spiked with cells of the MCF-7 line, and the cell mixture was labeled with anti-epithelial membrane antigen antibody and a magnetic colloid. The MCF-7 cells were selectively captured on a magnetic deposition substrate from the flowing leukocyte and MCF-7 cell mixture. RESULTS: The recovery of the MCF-7 cells from the original mixture ranged from 20% to 60%. The limit of detection of the MCF-7 cells was 10(-6) (n = 9). The morphology of the captured cancer cells was well preserved and comparable to that observed in cytospin smears. All deposited cells were located in a small area of 1.4 mm x 6 mm and could be quickly identified with an optical microscope following Wright's staining. CONCLUSIONS: This is a proof-of-principle study using a simplified model of rare cancer cells in a leukocyte mixture. The clinical relevance of the method will be tested in the future by extension to patient bone marrow samples and using antibody cocktails to increase specificity against the breast carcinoma cells.     

In situ analysis of changes in telomere size during replicative aging and cell transformation

Telomeres have been shown to gradually shorten during replicative aging in human somatic cells by Southern analysis. This study examines telomere shortening at the single cell level by fluorescence in situ hybridization (FISH). FISH and confocal microscopy of interphase human diploid fibroblasts (HDFs) demonstrate that telomeres are distributed throughout the nucleus with an interchromosomal heterogeneity in size. Analysis of HDFs at increasing population doubling levels shows a gradual increase in spot size, intensity, and detectability of telomeric signal. FISH of metaphase chromosomes prepared from young and old HDFs shows a heterogeneity in detection frequency for telomeres on chromosomes 1, 9, 15, and Y. The interchromosomal distribution of detection frequencies was similar for cells at early and late passage. The telomeric detection frequency for metaphase chromosomes also decreased with age. These observations suggest that telomeres shorten at similar rates in normal human somatic cels. T-antigen transformed HDFs near crisis contained telomere signals that were low compared to nontransformed HDFs. A large intracellular heterogeneity in telomere lengths was detected in two telomerase-negative cell lines compared to normal somatic cells and the telomerase-positive 293 cell line. Many telomerase-negative immortal cells had telomeric signals stronger than those in young HDFs, suggesting a different mechanism for telomere length regulation in telomerase-negative immortal cells. These studies provide an in situ demonstration of interchromosomal heterogeneity in telomere lengths. Furthermore, FISH is a reliable and sensitive method for detecting changes in telomere size at the single cell level.     

In vitro selection of aptamer S1 against MCF-7 human breast cancer cells

Breast cancer is the most common female cancer. However, the known effective specific biomarkers for breast cancer are still scarce. Abnormal membrane proteins serve as ideal biomarkers for disease diagnoses, therapeutics and prognosis. Thus aptamers (single-stranded oligonucleotide molecules) with molecular recognition properties can be used as efficient tools to sort cells based on differences in cell surface architecture between normal and tumor cells. In this study, we aimed to screen specific aptamer against MCF-7 human breast cancer cells. Cell-SELEX process was performed to isolate aptamers from a combinatorial single-stranded nucleic acid library that selectively targeting surface proteins of MCF-7 cells in contrast with MCF-10A human mammary epithelial cells. The process was repeated until the pool was enriched for sequences that specifically recognizing MCF-7 cells in monitoring by flow cytometry. Subsequently, the enriched pool was cloned into bacteria, and positive clones were sequenced to obtain individual sequences. Representative sequences were chemically synthesized and evaluated their binding affinities to MCF-7 cells. As a result, an aptamer S1 was finally identified to have high binding affinity with equilibrium dissociation constant (K_d) value of 29.9 ± 6.0 nM. FAM-labeled aptamer S1 induced fluorescence shift in MCF-7 cells but not in MCF-10A human mammary epithelial cells, or MDA-MB-453 and MDA-MB-231 human breast cancer cells. Furthermore, result of cell imaging observed from laser confocal fluorescence microscope showed that MCF-7 cells exhibited stronger fluorescence signal resulted from Cy5-labeled aptamer S1 than MCF-10A cells. The above findings suggested that S1 may be a specificity and selectivity aptamer for MCF-7 cells and useful for the breast cancer detection and diagnosis.     

X-ray enabled detection and eradication of circulating tumor cells with nanoparticles

The early detection and eradication of circulating tumor cells (CTCs) play an important role in cancer metastasis management. This paper describes a new nanoparticle-enabled technique for integrated enrichment, detection and killing of CTCs by using magnetic nanoparticles and bismuth nanoparticles, X-ray fluorescence spectrometry, and X-ray radiation. The nanoparticles are modified with tumor targeting agents and conjugated with tumor cells through folate receptors over-expressed on cancer cells. A permanent micro-magnet is used to collect CTCs suspended inside a flowing medium that contains phosphate buffered saline (PBS) or whole blood. The characteristic X-ray emissions from collected bismuth nanoparticles, upon excitation with collimated X-rays, are used to detect CTCs. Results show that the method is capable of selectively detecting CTCs at concentrations ranging from 100-100,000cells/mL in the buffer solution, with a detection limit of ～100CTCs/mL. Moreover, the dose of primary X-rays can be enhanced to kill the localized CTCs by radiation induced DNA damage, with minimal invasiveness, thus making in vivo personalized CTC management possible.     

DEVDase detection in intact apoptotic cells using the cell permeant fluorogenic substrate, (z-DEVD)2-cresyl violet

Using a bisubstituted caspase-3 target sequence: aspartate-glutamate-valine-aspartate, (z-DEVD)2 peptide derivative of the fluorophore, cresyl violet, we have obtained a cell permeant, fluorogenic, caspase substrate capable of detecting the site-specific presence of functionally active, caspase-3 and caspase-7 up-regulation within intact apoptotic cells. Addition of this substrate to induced and noninduced cell culture populations allows for the rapid site-specific detection of caspase up-regulation without the requirement for a wash step. We demonstrate here the use of (z-DEVD)2-cresyl violet substrate for the detection of apoptosis induction in Jurkat, THP-1, and MCF-7 cells using fluorescence microscopy and 96-well fluorescence plate reader analysis. Intracellular up-regulated DEVDase activity, which was clearly visible by fluorescence microscopy and 96-well fluorescence plate reader measurements, showed greater than 6-fold increases in fluorescence output in induced versus noninduced Jurkat cell samples. A simple fluorogenic substrate conversion method is demonstrated here for detecting apoptosis induction within intact living cells.     

Chemical sensing by a novel electrical oscillator: detection and quantitation of polysaccharides in concanavalin A solutions

Electrical oscillations across two platinum electrodes connected to an external circuit and immersed in HEPES buffer solutions of concanavalin A (Con A) were measured at various concentrations of mannan, starch and dextran. The frequency of oscillations was found to change almost linearly with the logarithm of the concentration of mannan between 10(-4) and 1 w/v%; whereas the frequency remained nearly constant with dextran. With starch it slightly increased as the concentration increased. This method was suggested to be useful for quantitative and selective measurement of antigen-antibody reaction and also for detection of various cells with the specific binding site such as cancer cells.     

In situ localization of alkaline phosphatase activity in tumor cells by an aggregation-induced emission fluorophore-based probes

In situ detection of certain specific enzyme activities in cells is deeply attached to tumor diagnosis. Conventional enzyme-responsive fluorescent probes have difficulty detecting targeted enzymes in situ in cells due to the low detection accuracy caused by the spread of fluorescence probes. In order to solve this problem, we have designed and synthesized an enzyme-responsive, water-soluble fluorescent probe with AIE characteristics, which could aggregate and precipitate to produce in situ fluorescence when reacting with the targeted enzyme in cells. The AIE fluorophore (TPEQH) was utilized to design the enzyme-responsive, fluorescent probe (TPEQHA) by introducing a phosphate group on to it, which could be specifically decomposed by the targeted enzyme, namely alkaline phosphatase (ALP). In tumor cells, TPEQH was highly produced due to the interaction of phosphate on the TPEQHA and the overexpressed ALP. Water-insoluble TPEQH then precipitated and release fluorescence in situ, thereby successfully detecting the ALP. Furthermore, the expression level of ALP could be determined by the fluorescence intensity of TPEQH with higher accuracy due to the inhibition of TPEQH leak, which demonstrated a potential application of in suit ALP detection in both clinical diagnosis and scientific research of tumor.     

Detection of CD133-marked cancer stem cells by surface plasmon resonance: Its application in leukemia patients

Here, we reported the development of a label-free and real-time surface plasmon resonance (SPR) based biosensor for cancer stem cells (CSCs) detection using cell surface biomarker; CD133. The fabricated biosensor was used for detection of this marker in some acute myeloid leukemia (AML) patients and the results were compared with those obtained from flow cytometry (FC) method. CD133 antibody was immobilized on the gold chip surface via EDC/NHS coupling method and binding of the candidate cells to the modified gold sensor surface was monitored after isolation of mononuclear cells from bone marrow of the patients. The method was validated in terms of various parameters such as CD133- antibody concentration and cell density. The CD133-marked cells were investigated in seven AML patients. All SPR results were compared with those obtained from FC method. A very good correlation (R² = 0.96) was obtained between SPR and FC responses related to CD133-marked cells densities. In conclusion, in this study, a label-free and real-time SPR cytometry method was developed to detect CD133 and it was successfully applied to follow this cancer stem cell biomarker in AML patients.     

Messenger RNA quantification after fluorescence activated cell sorting using intracellular antigens

Recent studies using stem cells or cancer stem cells have revealed the importance of detecting minor populations of cells in blood or tissue and analyzing their biological characteristics. The only possible method for carrying out such procedures is fluorescence activated cell sorting (FACS). However, FACS has the following limitations. First, cells without an appropriate cell surface marker cannot be sorted. Second, the cells have to be kept alive during the sorting process in order to analyze their biological characteristics. If an intracellular antigen that was specific to a particular cell type could be stained with a florescent dye and then the cells can be sorted without causing RNA degradation, a more simple and universal method for sorting and analyzing cells with a specific gene expression pattern could be established since the biological characteristics of the sorted cells could then be determined by analyzing their gene expression profile. In this study, we established a basic protocol for messenger RNA quantification after FACS (FACS-mQ) targeting intracellular antigens. This method can be used for the detection and analysis of stem cells or cancer stem cells in various tissues.