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Flow Cytometry


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  • Using flow cytometry to compare the dynamics of photoreceptor outer segment phagocytosis in iPS-derived RPE cells. 22871841

    Retinal pigment epithelium (RPE) autologous grafts can be readily derived from induced pluripotent stem (iPS) cells. It is critical to stringently characterize iPS-RPE using standardized and quantifiable methods to be confident that they are safe and adequate replacements for diseased RPE before utilizing them in clinical settings. One important and required function is that the iPS-RPE phagocytose photoreceptor outer segments (POS).We developed a flow cytometry-based assay to monitor binding and internalization of FITC labeled POS by ARPE-19, human fetal RPE (hfRPE), and two types of iPS-RPE. Expression and density of α(v)β₅ integrin, CD36, and MerTK receptors, which are required for phagocytosis, were compared.Trypsinization of treated RPE cells results in the release of bound POS. The number of freed POS, the percentage of cells that internalized POS, the brightness of the FITC signal from the cells, and the surface density of the phagocytosis receptors on single RPE cells were measured using flow cytometry. These assays reveal that receptor density is dynamic during differentiation and this can affect the binding and internalization dynamics of the RPE cells. Highly differentiated iPS-RPE phagocytose POS more efficiently than hfRPE.Caution should be exercised to not use RPE grafts until demonstrating that they are fully functional. The density of the phagocytosis receptors is dynamic and may be used as a predictor for how well the iPS-RPE cells will function in vivo. The phagocytosis dynamics observed between iPS-RPE and primary RPE is very encouraging and adds to mounting evidence that iPS-RPE may be a viable replacement for dysfunctional or dying RPE in human patients.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere

  • Flow cytometry analysis: a quantitative method for collagen VI deficiency screening. 22075033

    Mutations in COL6A1, COL6A2 and COL6A3 genes result in collagen VI myopathies: Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM) and intermediate phenotypes. At present, none of the existing diagnostic techniques for evaluating collagen VI expression is quantitative, and the detection of subtle changes in collagen VI expression remains challenging. We investigated flow cytometry analysis as a means of quantitatively measuring collagen VI in primary fibroblasts and compared this method with the standard method of fibroblast collagen VI immunohistochemical analysis. Eight UCMD and five BM molecularly confirmed patients were studied and compared to five controls. Flow cytometry analysis consistently detected a reduction of collagen VI of at least 60% in all UCMD cases. In BM cases the levels of collagen VI were variable but on average 20% less than controls. Flow cytometry analysis provides an alternative method for screening for collagen VI deficiency at the protein level in a quantitative, time and cost-effective manner.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    MAB1944
    Produktbezeichnung:
    Anti-Collagen Type VI Antibody, clone 3C4

  • Evaluation by flow cytometry of antibody-dependent enhancement (ADE) of dengue infection by sera from Thai children immunized with a live-attenuated tetravalent dengue va ... 15315835

    Sera from Thai children immunized with a live-attenuated tetravalent dengue virus vaccine or from naturally infected age-matched site-control subjects were examined for immune enhancement capacity by a highly reproducible flow cytometric assay in Fc receptor-bearing K562 human cells. None of the sera under study corresponded to cases of severe dengue disease. In parallel assays employing each dengue virus serotype, we found no or only minimal antibody-dependent enhancement (ADE) when sera from vaccinated or control subjects were used at a low serum dilution [1/12] that approximated the in vivo condition. Among sera that exhibited homotypic neutralizing antibody activity against DV1-3, the level correlated with absence of ADE or infection with the respective serotype. Similarly, a broad heterotypic neutralizing antibody response that included all four serotypes was linked to complete absence of K562 cell infection. In contrast, at higher serum dilutions a correlation between breadth of antibody response and heightened immune enhancement emerged, a pattern identical to that observed among control subjects. These findings support the use of live dengue vaccines and protocols that induce broad serotype-specific neutralizing antibody responses, but they also suggest that clinically relevant immune enhancement may not be likely if this is not uniformly achieved after the first immunization.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    MAB8703
    Produktbezeichnung:
    Anti-Dengue Virus Type III Antibody, clone 5D4-11

  • Validation of a flow cytometry based G(2)M delay cell cycle assay for use in evaluating the pharmacodynamic response to Aurora A inhibition. 20887727

    Pharmacodynamic assays are important aspects for understanding molecularly targeted anticancer agents to investigate the relationship between drug concentration (pharmacokinetics) and drug "effect" or biological activity. As new drug entities are developed that affect DNA cell cycle, a pharmacodynamic assay which measures cell cycle perturbation would be a valuable clinical trial tool. During recent years, flow cytometry has established itself as a useful method to determine the relative nuclear DNA content and percentage of cycling cells of biological specimens. However to date, the analytical validation of cytometry based assays is limited and there is no suitable guidance for method validation of flow cytometry based cell cycle assays. Here we report the validation of a flow cytometry based cell cycle G(2)/M delay assay for use in evaluating the effect of investigational drug MLN8237, a small molecule inhibitor of a mitotic kinase Aurora A, for clinical trial use. The assay method was validated by examining assay robustness, repeatability, reproducibility, precision, and determining the cutoff for a true drug effect based on biostatistical analysis models. Experimental results show that the intra-assay repeatability was less than 20% with an intra-donor variability of less than 40%. The robustness of the assay was less than 30%. Since this is an ex-vivo stimulation assay, variability parameters were expected to be higher. Based on biostatistical modeling, an absolute change in %G(2)M of 5.2% (95% CI) was needed in order to detect a true drug effect. Overall, the assay demonstrated acceptable variability to warrant further in vivo testing.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    16-202A
    Produktbezeichnung:
    Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301, FITC conjugate

  • Use of time-saving flow cytometry for rapid determination of resistance of human cytomegalovirus to ganciclovir. 16207954

    There are two ways to assess the susceptibility of human cytomegalovirus (HCMV) to ganciclovir (GCV): one is a genotypic test that detects resistance-related mutations and the other is a phenotypic test that actually assesses susceptibility. The advantages of genotyping the UL 97 gene are its rapidity and accuracy. However, to detect novel mutations or mutations affecting the UL 54 DNA polymerase, a phenotypic test such as the plaque reduction assay (PRA) is also required. To avoid the shortcomings of PRA such as its time-consuming nature and labor-intensiveness, we developed a time-saving fluorescence-activated cell sorting (TS-FACS) technique. We obtained a GCV 50% inhibitory concentration (IC(50)) from five clinical isolates and an HCMV laboratory strain (AD169) and compared the results with those from the PRA. The laboratory strain and three clinical isolates were sensitive to GCV. Although there was a minor discrepancy in the case of one of the three isolates, the GCV IC(50) values obtained by TS-FACS analysis correlated well with the results of the PRA. The remaining two isolates were resistant to GCV; one was GCV resistant due to the mutation M 460 V, and the GCV IC(50) results obtained by TS-FACS analysis and by PRA were also comparable. The advantages of TS-FACS analysis are the shorter time required, the possibility of automation, and its comparability to PRA, considered the gold standard. Thus, TS-FACS analysis may be useful as an alternative to PRA in the clinic.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    5090
    Produktbezeichnung:

  • Optimized flow cytometry isolation of murine spermatocytes. 24664803

    Meiotic prophase I (MPI), is an initial stage of meiosis characterized by intricate homologous chromosome interactions, synapsis, and DNA recombination. These processes depend on the complex, but poorly understood early MPI events of homologous chromosome search, alignment, and pairing. Detailed molecular investigation of these early events requires isolation of individual MPI substages. Enrichment for Pachytene (P) and Diplotene (D) substages of late MPI was previously accomplished using flow cytometry. However, separation of early MPI spermatocytes, specifically, of Leptotene (L) and Zygotene (Z) substages, has been a challenge due to these cells' similar characteristics. In this report, we describe an optimized Hoechst-33342 (Hoechst)-based flow cytometry approach for isolating individual MPI populations from adult mouse testis. We get significant enrichment for individual L and Z spermatocytes, previously inseparable from each other, and optimize the isolation of other MPI substages. Our flow cytometry approach is a combination of three optimized strategies. The first is optimization of testis dissociation protocol that yields more consistent and reproducible testicular single cell suspension. The second involves optimization of flow cytometric gating protocol where a critical addition to the standard protocol for cell discrimination based on Hoechst fluorescence, involves a back-gating technique based on light scattering parameters. This step specifies selection of individual MPI substages. The third, is an addition of DNA content restriction to the gating protocol to minimize contamination from non-meiotic cells. Finally, we confirm significant enrichment of high-purity Preleptotene (PreL), L, Z, P, and D MPI spermatocytes using stage-specific marker distribution. The technique will facilitate understanding of the molecular events underlying MPI.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    05-636
    Produktbezeichnung:
    Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301

  • High-resolution multiparameter DNA flow cytometry for the detection and sorting of tumor and stromal subpopulations from paraffin-embedded tissues. 19816924

    This unit contains a detailed protocol for the simultaneous flow cytometric measurement of tumor cells, stromal cells, and DNA content of formalin-fixed, paraffin-embedded (FFPE) tissues. The vimentin-positive stromal cell fraction can be used as an internal reference for DNA content assessments. This allows clear detection of keratin-positive tumor cells with a DNA index lower than 1.0 and of keratin-positive tumor cells with a DNA close to 1.0 in overall DNA aneuploid samples, thus improved DNA ploidy assessment in FFPE carcinomas. Furthermore, the protocol is useful for studying molecular genetic alterations and intratumor heterogeneity in archival FFPE samples. Keratin-positive tumor cell fractions can be flow-sorted for further molecular genetic analysis, while DNA from the sorted vimentin-positive stromal cells can serve as a reference when normal tissue of the patient is not available.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    MAB3412
    Produktbezeichnung:
    Anti-Cytokeratin AE1/AE3 Antibody, recognizes acidic & basic cytokeratins, clone AE1/AE3

  • Comparison of diagnostic accuracy of microscopy and flow cytometry in evaluating N-methyl-D-aspartate receptor antibodies in serum using a live cell-based assay. 25815887

    N-methyl-D-aspartate receptor (NMDAR) encephalitis is an autoimmune neurological disease, diagnosed by a specific autoantibody against NMDAR. Antibody testing using commercially available cell-based assays (CBA) or immunohistochemistry on rat brain tissue has proven high specificity and sensitivity. Here we compare an immunofluorescence live CBA to a flow cytometry (FACS) based assay to detect NMDAR antibodies by their binding to the surface of HEK293A cells functionally expressing NMDAR. Both assays were first established using a discovery group of 76 individuals and then validated in a group of 32 patients in a blinded manner. In the CBA, 23 of 23 patients with NMDAR encephalitis were positive for NMDAR antibodies and 0 of 85 controls (32 healthy controls and 53 patients with other neurological diseases), resulting in a sensitivity and specificity of 100% (95% confidence intervals (CI) 85.1-100.0 and 95.7-100.0, respectively). The FACS based assay detected NMDAR antibodies in 20 of 23 patients and in 0 of 85 controls. Therefore, with an equally high specificity (95% CI 95.7-100.0) the sensitivity of the FACS based assay was 87% (95% CI 66.4-97.2). Comparing antibody titers from CBA with delta median fluorescence intensities from FACS showed a high concordance (kappa = 0.943, pless than 0.0001) and correlation (r = 0.697, pless than 0.0001). In conclusion, evaluation of the FACS based assay revealed a lower sensitivity and high inter-assay variation, making the CBA a more reliable detection method.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere
    Produktbezeichnung:
    Mehrere

  • Detection of p53 dysfunction by flow cytometry in chronic lymphocytic leukaemia. 15521919

    In chronic lymphocytic leukaemia (CLL) cells, functional impairment of the p53 pathway is detectable by Western blotting as impaired up-regulation of p21 (a transcriptional target of p53) in response to ionizing radiation (IR). The type A defect is characterized by baseline p53 overexpression and is associated with TP53 mutation. The type B defect is characterized by impaired IR-induced p53 up-regulation and is associated with inactivation of the ataxia telangiectasia-mutated gene (ATM). Both abnormalities are strongly associated with adverse clinical outcome. In the present study, flow cytometry was found to be an effective alternative to Western blotting in the detection of p53 dysfunction in CLL.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    MABE325
    Produktbezeichnung:
    Anti-p21WAF1 Antibody, clone EA10