DAPI Fluorescence in Nuclei Isolated from Tumors

Krishan, Awtar; Dandekar, Payal

doi:10.1369/jhc.4b6563.2005

Cited by 22 publications

(12 citation statements)

References 10 publications

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“…Then the cells were incubated with FITC-conjugated goat anti-mouse antibody (Calbiochem, USA) in PBS containing 1% BSA for 1 h at 37 °C and then washed twice in PBS containing 1% BSA. The immunofluorescently labeled cells were also labeled with 10 mg/L of 4′-6-diamidino-2-phenylindole (DAPI, Sigma USA) in PBS to visualize the nuclei [31]. Finally, the coverslips were mounted onto slides with a mounting medium (80% glycerol) and analyzed using a confocal laser scanning microscope (LSM 510 META Carl Zeiss).…”

Section: Methodsmentioning

confidence: 99%

Tunicamycin Depresses P-Glycoprotein Glycosylation Without an Effect on Its Membrane Localization and Drug Efflux Activity in L1210 Cells

Šereš

Cholujová

Bubencíkova

et al. 2011

IJMS

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P-glycoprotein (P-gp), also known as ABCB1, is a member of the ABC transporter family of proteins. P-gp is an ATP-dependent drug efflux pump that is localized to the plasma membrane of mammalian cells and confers multidrug resistance in neoplastic cells. P-gp is a 140-kDa polypeptide that is glycosylated to a final molecular weight of 170 kDa. Our experimental model used two variants of L1210 cells in which overexpression of P-gp was achieved: either by adaptation of parental cells (S) to vincristine (R) or by transfection with the human gene encoding P-gp (T). R and T cells were found to differ from S cells in transglycosylation reactions in our recent studies. The effects of tunicamycin on glycosylation, drug efflux activity and cellular localization of P-gp in R and T cells were examined in the present study. Treatment with tunicamycin caused less concentration-dependent cellular damage to R and T cells compared with S cells. Tunicamycin inhibited P-gp N-glycosylation in both of the P-gp-positive cells. However, tunicamycin treatment did not alter either the P-gp cellular localization to the plasma membrane or the P-gp transport activity. The present paper brings evidence that independently on the mode of P-gp expression (selection with drugs or transfection with a gene encoding P-gp) in L1210 cells, tunicamycin induces inhibition of N-glycosylation of this protein, without altering its function as plasma membrane drug efflux pump.

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Section: Methodsmentioning

confidence: 99%

Tunicamycin Depresses P-Glycoprotein Glycosylation Without an Effect on Its Membrane Localization and Drug Efflux Activity in L1210 Cells

Šereš

Cholujová

Bubencíkova

et al. 2011

IJMS

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“…In low concentrations, like the one used in these experiments, a strong fluorescence signal is only observable in cells whose membrane were perforated, i.e. in dead and/or optoporated cells, as DAPI passes very slowly through intact cellular membranes 23 . Calcein AM is originally a non-fluorescent compound which however, emits green-fluorescence after hydrolysis by intracellular esterase in living cells.…”

Section: Methodsmentioning

confidence: 99%

Software-aided automatic laser optoporation and transfection of cells

Breunig

Uchugonova

Batista

et al. 2015

Sci Rep

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Optoporation, the permeabilization of a cell membrane by laser pulses, has emerged as a powerful non-invasive and highly efficient technique to induce transfection of cells. However, the usual tedious manual targeting of individual cells significantly limits the addressable cell number. To overcome this limitation, we present an experimental setup with custom-made software control, for computer-automated cell optoporation. The software evaluates the image contrast of cell contours, automatically designates cell locations for laser illumination, centres those locations in the laser focus, and executes the illumination. By software-controlled meandering of the sample stage, in principle all cells in a typical cell culture dish can be targeted without further user interaction. The automation allows for a significant increase in the number of treatable cells compared to a manual approach. For a laser illumination duration of 100 ms, 7-8 positions on different cells can be targeted every second inside the area of the microscope field of view. The experimental capabilities of the setup are illustrated in experiments with Chinese hamster ovary cells. Furthermore, the influence of laser power is discussed, with mention on post-treatment cell survival and optoporation-efficiency rates.

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“…Details of the instrumentation, optical configuration, and our staining protocols have been reported earlier. [7][8][9][13][14][15] Software programs, ModFit (Verity Software House, Topsham, ME) and WinMDI 2.8 (downloaded from http://facs.scripps.edu/) were used to analyze the list mode data and calculate DNA Index (DI).…”

Section: Methodsmentioning

confidence: 99%

Detection of tumor cells in body cavity fluids by flow cytometric and immunocytochemical analysis

et al. 2006

Self Cite

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Measurement of electronic volume versus DNA content of nuclei can be used to discriminate between normal and malignant cells. Epithelial membrane antigen immunocytochemistry (EMA-ICC), a helpful ancillary test in body cavity fluids, is not universally accurate for detecting malignancy in effusions. The current study was undertaken to determine if multiparametric flow cytometry (based on simultaneous analysis of light scatter, nuclear volume, DNA, and nuclear protein content) in combination with (EMA-ICC) could be used for the detection of malignant cells in peritoneal and pleural fluids. We studied 130 body cavity fluids (68 peritoneal and 62 pleural fluids) by conventional cytology and multiparametric laser flow cytometry. EMA-ICC was performed using EMA antibodies and L-SAB detection system (DakoCytomation, Carpinteria, CA). EMA-ICC had significantly higher sensitivity than conventional cytology (79% versus 59%, P = 0.016) and ploidy (79% versus 38%, P = 0.001). Cytology had significantly higher specificity than ploidy (97% versus 82%, P = 0.012). The differences in specificity between EMA-ICC and ploidy (87% versus 82%, P= 0.607) or EMA-ICC and cytology (87% versus 97%, P = 0.109) were not statistically significant. However, assuming serial testing, sensitivity increased significantly for the combinations of cytology and EMA-ICC (79.4%, P = 0.016) and cytology and ploidy (73.5%, P = 0.004) as compared to cytology alone (58.8%). Also, the combination of cytology and ploidy had a higher sensitivity than ploidy alone (73% versus 38%, P < 0.0001). However, the sensitivity associated with the three tests used in serial (85.3%) was not significantly different from the sensitivities corresponding to the combination of cytology and EMA-ICC (79%) or cytology and ploidy (73%). Multiparametric flow cytometry utilizing high resolution DNA, nuclear volume, protein measurement, and ICC, in combination with cytomorphology, may be a valuable tool for rapid identification of malignant cells in body cavity fluids.

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DAPI Fluorescence in Nuclei Isolated from Tumors

Cited by 22 publications

References 10 publications

Tunicamycin Depresses P-Glycoprotein Glycosylation Without an Effect on Its Membrane Localization and Drug Efflux Activity in L1210 Cells

Tunicamycin Depresses P-Glycoprotein Glycosylation Without an Effect on Its Membrane Localization and Drug Efflux Activity in L1210 Cells

Software-aided automatic laser optoporation and transfection of cells

Detection of tumor cells in body cavity fluids by flow cytometric and immunocytochemical analysis

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