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EN
One of the most frequently implemented processes in the water treatment technology is filtration through a biosorption bed. The techniques based on biochemical processes involving bacteria result in obtaining high quality of water. There are a number of different materials used as the filler material for biological filters. Carbon deposits are the most popular, due to their high effectiveness. The problem with the use of this process is the leaching of microorganisms from the biofilm and the biological stability of water thus obtained. There is a need to develop quick methods to assess the microbiological quality of this water. Modern techniques for determining the amount of microorganisms, such as flow cytometry and luminometry may be the right tools. The water collected for testing came from the Water Treatment Station located in the Podkarpackie voivodeship. The microbiological tests carried out in the analyzed water samples collected after the filtration process on granular activated carbon. Both traditional culture method and modern techniques used to determine the number of microorganisms (flow cytometry, luminometric ATP assay) demonstrated an increase in the number of microorganisms in the examined waters (in the water after the filtration process and in the water introduced into the water supply network) after the incubation process for 3 and 7 days at 15 and 22°C.
EN
Introduction. Treatment and diagnostic process in solid tumors like lung cancer are still based on invasive methods such as bronchoscopy, solid biopsy et cetera. One of the less invasive methods is a proposed “liquid biopsy” which is based on capturing of tumor cells circulating in the blood. Aim. The aim of the study was to standardize conditions and to assess the sensitivity of the identification of circulating tumor cells (CTCs) with the use of flow cytometry and qRT-PCR. Material and methods. In the first model of CTCs, cells from the A549 lung cancer cell line were suspended in 1 ml of healthy donors’ blood in 5 spikes increasingly: 0, 10, 50, 100 and 200 and the cells were detected in flow cytometer. In the second model, cells from the A549 and H1975 lung cancer cell lines were used. Spikes were prepared as in the first model, but cells were suspended in 400 µl of healthy donors’ blood and were detected with the use of qRT-PCR. Results. An increasing number of detected cytokeratin positive events from the 1st spike “0” to the last one - “200” was observed by flow cytometry. Median value in the negative control was 0 false positive cells. In tubes from “10” to “200” the median was 5, 43.5, 58 and 78, respectively. Mean sensitivity of flow cytometry was 63.79%. In qRT-PCR, correlation between increasing number of sorted cells in several spikes and the level of mRNA expression for KRT19 gene was not observed. Conclusion. Commonly available methods like flow cytometry and qRT-PCR seem to be attractive solutions for CTCs detection, but they need pre-enrichment procedures and standardization
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Flow cytometry is a technology that simultaneously counts and then examines multiple physical and/or chemical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light. The properties measured include a particle’s relative size, relative granularity or internal complexity, and relative fluorescence intensity. These characteristics are determined using an optical-to-electronic coupling system that records how the cell scatters incident laser light and emits fluorescence. One of the most significant applications is immunophenotyping of cells - the most important tool in diagnosis and monitoring haematological disorders, such as acute and chronic leukemia, lymphoma, monoclonal gammopathy, myelodisplastic and myeloproliferative diseases.
EN
Growing areas of sealed surfaces, rising water needs due to industry development, increasing populations, and climatic changes affect precipitation patterns and form a vision of the future in which meteoric water storage may become almost an obligatory activity. The aim of this paper was to identify the amounts and, to some degree, the quality of microorganisms present in rainwater collected from different types of rooftops of utility buildings in the spring-summer season. Apart from the classic culture plate method complemented by flow cytometry. The results of performed analyses explicitly show that rainwater collected from rooftops and directly from air prove to be microbially contaminated to a substantial degree, which includes pathogenic coliforms and faecal streptococci. Waters collected after dry periods also contained bacteria like Clostridium perfingens. The findings rule out the possibility of using rainwater collected from roof surfaces of utility buildings before its treatment.
EN
Structural motifs found in naturally occurring compounds are frequently used by researchers to develop novel synthetic drug candidates. Some of these new agents are hybrid molecules which are designed through a concept of combining more than one functional element. In this report, anticancer activity of new synthetic molecular hybrids, substituted 3-diethoxyphosphorylnaphtho[2,3-b]furan-4,9-diones and 3-diethoxyphosphorylbenzo[f]indole-4,9-diones, which integrate natural 1,4-naphtalenedione scaffold, present in several anticancer agents, with pharmacophoric phosphonate moiety, were tested against hepatocellular cell line HepG2. Cytotoxicity was examined using MTT assay. Two most potent compounds, furandione 8a and benzoindoldione 12a, which reduced the number of viable HepG2 cells with the IC50 values of 4.13 µM and 5.9 µM, respectively, were selected for further research. These compounds decreased the mRNA expression levels of several genes: Bcl-2, angiogenic vascular endothelial growth factor (VEGF), c-Fos, caspase-8 and increased the expression of Bax, caspase-3 and -9, c-Jun, p21, p53, as determined by quantitative real-time PCR. The ability of these compounds to induce apoptosis and DNA damage was studied by flow cytometry. The obtained data showed that the new compounds inhibited cell viability by increasing apoptosis and decreasing angiogenesis. Compound 8a was a much stronger apoptosis inducer as compared with 12a and strongly activated the intrinsic pathway of apoptosis, associated with the loss of mitochondrial membrane potential and changes in Bax/Bcl-2 ratio. These findings show that the synthetic hybrids combining 1,4-naphthalenedione system and phosphonic acid moiety display potential to be further explored in the development of new anticancer agents.
EN
(E)-2'-deoxy-2'-(fluoromethylene)-cytidine (FMdC), a deoxycytidine analog displaying a very high toxicity toward a variety of solid tumor cell lines and xenografts, is activated intracellularly by deoxycytidine kinase (dCK). We have compared cytotoxicity of FMdC towards a human promyeolocytic leukemia line HL-60 and a human colorectal carcinoma line COLO-205. Despite dCK activity being by far the highest in cells of lymphoid origin, the effects of FMdC were detectable at the lowest drug concentration only in a solid tumor cell line, and at higher concentrations they were qualitatively similar in the two tumor lines (increased cell protein content, cell cycle block and apoptosis). Apparently, low dCK activity in solid tumor cells sufficiently activates FMdC to yield cytotoxic effects, while high dCK activity in leukemia cells does not increase its cytotoxicity.
EN
Appropriate validation of any bioassay to be used in the characterization of biological products is critical. In this study, we report a validation study of a flow cytometry-based assay to measure the complement-dependent cytotoxicity (CDC) of a biosimilar candidate monoclonal antibody (Mab) directed to CD20 antigen, as indicative of its biological activity. The assay was validated by examining: assay robustness, specificity, repeatability and intermediate precision. It demonstrated to be robust for all factors evaluated. It also showed a high level of specificity and was found to be free of interference through the validation process. The degree of precision (Cvs < 7%) obtained in this study was satisfactory. The presented work demonstrated that a flow cytometry-based cytotoxicity assay is a suitable method in the lot to lot quality control monitoring of the culture supernatant and an active pharmaceutical ingredient of 1B8 Mab.
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Content available remote Monitoring of T-cell acute lymphoblastic leukemia by flow cytometry
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EN
Minimal residual disease (MRD) predicts the outcome of acute lymphoblastic leukemia (ALL). Flow cytometry (FC) is one of the most sensitive and most applicable methods for MRD diagnostics, but there is still no agreement on the “gold standard” of the method. We tried to optimize flow cytometric MRD detection in T-ALL. Fourteen adults and 11 children with T-ALL and 12 normal bone marrow (BM) donors were enrolled in the study. We found that the most common phenotypic aberrations in T-ALL were TdT and CD99 coexpression on T-cells in BM. Therefore for MRD detection we developed a limited four-color marker panel (TdT/CD7/cCD3/CD19 and CD99/CD7/cCD3/CD2) and a standard analysis strategy. This assay was evaluated on BM of healthy controls. Less than 0.01% TdT+ or CD99 bright T-cells were found in normal BM. MRD was detected in 9 adult patients and 1 child at different time-points of treatment. The average TdT and CD99 mean fluorescence intensity (MFI) value of residual blasts fluctuated during therapy, but it still remained higher than MFI of normal T-cells. Our established MRD detection method differentiated leukemic lymphoblasts with sensitivity in the range of 0.01% and did not give any false positive results in normal BM.
EN
Flow cytometry is a method of identification biological agents that has various applications. It has been applied for identifying many types of antigens in various materials, including environmental samples. Recently it has been noticed that this method could be used for molecular detection of biological agents. The purpose of this work was to apply flow cytometry with nested-PCR for the molecular identification of B. anthracis. Paramagnetic streptavidin-coated beads were used to capture the resulting fluorophore-labeled sequences. The results show that flow cytometry can be successfully used to detect specific fluorescein- dUTP and a biotin marked sequences.
PL
Cytometria przepływowa jest metodą identyfikacji czynników biologicznych o wielu zastosowaniach. Używano ją do wykrywania różnego rodzaju antygenów w różnych typach próbek także środowiskowych. Ostatnio metody cytometryczne zaczęto stosować w diagnostyce molekularnej czynników biologicznych. Celem tej pracy było zastosowanie metody cytometrii przepływowej i zagnieżdżonej reakcji łańcuchowej polimerazy DNA (nested -PCR) w diagnostyce molekularnej Bacillus anthracis. Użyto paramagnetyczne mikrokulki opłaszczone streptawidyną na których immobilizowany był wyznakowany fluorochromami produkt PCR. Wyniki wykazały, że metoda cytometrii przypływowej może służyć do wykrywania specyficznych sekwencji wyznakowanych fluoresceinąi biotyną.
EN
Lymphocytes of haematologically and serologically EBB-positive cows were isolated and incubated with CD4, CD8, Cdllc and alpha bovine IgM markers, produced by Int. ILRAD Peter Maesseus. The cells were analysed immediately following fluorescein marking, in flow cytometer (FACS Calibur - Becton Dickinson). The number of proliferating B lymphocytes with increased IgM marker expression was high, testifying to early infection of the cattle with bovine leukemia virus (BLV). The expression of CD4 and CD8 markers in Th and Tc lymphocytes in leukemic cows was more pronounced than in healthy animals. The changes were accompanied by a decreased production of natural killer (NK) cells.
EN
In this work evaluation of usefulness of monoclonal antibodies (MAbs) CCH2 and AAC10 directed against early - pUL44(DB52) and late - ppUL83(pp65) CMV antigens, utilized in Department of Virology, NIH for routine diagnosis of CMV infection by shell vial and pp65 antigenemia assay, for determination of CMV antigens by flow cytometry in human leucocytes, isolated, infected and cultivated in vitro was presented.
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