Fluorescent DNA Stain Research Articles

Detection of mycoplasmas in cell cultures by PCR: a one year study

Over a one year period, 372 different cell cultures were received for the detection of mycoplasmas. PCR analysis of the 16SS rRNA gene was compared to DNA fluorescence staining by DAPI (4′,6-diamidine-2-phenylindole dihydrochloride). 278 samples (75%) were found to be negative using both methods, 86 samples (23%) were found positive with the PCR and the DAPI staining method, and 6 samples (2%) were found to be infected with other bacteria. The 86 positive samples were also subjected to specific amplification in order to identify the species. Only two of them (3.5%) could not be identified. In conclusion, PCR is a rapid and reliable method for detecting and identifying mycoplasmas in tissue cultures.

Identification of hair cell progenitors and intermitotic migration of their nuclei in the normal and regenerating avian inner ear

Postembryonic production of sensory hair cells occurs in both normal and aminoglycoside-damaged avian inner ears. The cellular source and mechanism that results in new differentiated hair cells were investigated in the avian vestibular epithelia using three distinct cell-cycle-specific labeling methods to identify proliferating sensory epithelial cells. First, immunocytochemical detection of the proliferating cell nuclear antigen, an auxiliary protein of DNA polymerase, allowed labeling of cells in late G1, S, and early G2 phases of the cell cycle. Second, a pulse-fix tritiated thymidine autoradiographic protocol was used to identify cells in S phase of the cell cycle. Finally, Hoechst 33342, a fluorescent DNA stain, was used to identify epithelial cells in mitosis. The distribution of cells active in the cell cycle within the normal and ototoxin-damaged vestibular epithelium suggests that supporting cells within the sensory epithelia are the cellular precursors to the regenerated hair cells. Differences between the proliferation marker densities in control and damaged end organs indicate that the upregulation of mitotic activity observed after streptomycin treatment is due primarily to an increase in the number of dividing progenitor cells. The differences between the extent of ototoxic damage and the level of reparative proliferative response suggest a generalized stimulus, such as a soluble chemical factor, plays a role in initiating regeneration. Finally, after DNA replication is initiated, progenitor cell nuclei migrate from their original location close to the basement membrane to the lumenal surface, where cell division occurs. This pattern of intermitotic nuclear migration is analogous to that observed in the developing inner ear and neural epithelium.

Comparison of Three Methods for the Detection of Mycoplasms in Cell Cultures

ABSTRACTA total of 33 cell cultures was checked for mycoplasma contamination by microbiological culture method, fluorescent DNA-staining (Hoechst 33258) and hybridization assay. One third of the assayed cultures was contaminated. The sensitivity of the direct culture method was approximately 102 CFU/ml and that of the remaining methods 10s CFU/ml.

Formation of Stable Ustilago hordei Diploids

Ustilago hordei is a fungal pathogen of barley that causes covered smut disease. During asexual growth, this fungus reproduces as haploid uninucleate budding yeastlike cells (sporidia) that are nonpathogenic. Mating of sporidia, which is controlled by a single mating-type locus with two alleles, results in the formation of pathogenic dikaryotic mycelia. When sporidia of the opposite mating type with nonallelic auxotrophic mutations were plated together on minimal medium, yeastlike colonies grew at a frequency of 1 x 10 -6 . These cultures formed mycelia on charcoal agar, confirming the presence of both mating-type genes. These cells had a single nucleus, as determined by fluorescence microscopy, and protoplasts had a volume approximately twice that of protoplasts derived from haploid cells. Flow cytometry of haploid and suspected diploid cells treated with the fluorescent DNA stain propidium iodide showed that the diploids had roughly twice the DNA content of haploid cells. Pulsed field gel elect...

Specifity and sensitivity of polymerase chain reaction (PCR) in comparison with other methods for the detection of mycoplasma contamination in cell lines

The polymerase chain reaction (PCR) amplification was used for the detection of mycoplasma contamination in 42 continuous cell lines. Using the microbiological cultivation on agar as the reference method, 29 cell lines were regarded as positive and 13 cell lines as negative. The double-step PCR analysis employed nested primers that anneal to gene sequences coding for the evolutionarily conserved 16 S rRNA of some 25 different mycoplasma species (including the ones most commonly found in cell cultures). In terms of the positivity or negativity of mycoplasma infection the results were identical for the agar assay and PCR amplification. All positive cell lines displayed distinct, unequivocal, objectively discernible bands on agarose gels while the non-infected specimens showed no DNA amplification. A simultaneously performed comparison with four other commonly used detection methods (DNA-RNA hybridization in solution, DAPI DNA fluorescence staining, immunostaining with a monoclonal antibody and an ELISA) showed that PCR produced significantly less false-negative or false-positive results than all the other methods. Furthermore, in dilution experiments, PCR correctly detected the infecting mycoplasmas at the lowest level of 1/10 4 whereas the other assays were less sensitive. It is concluded that double-step PCR employing nested primers is superior to other mycoplasma detection methods in many respects: simplicity and speed, high specificity and extreme sensitivity, objectivity and accuracy.

Post-translational processing of atrial natriuretic factor by adult rat atrial cardiocytes in culture

Post-translational processing of the cardiac polypeptide hormone atrial natriuretic factor (ANF) was studied using primary cultures of cardiocytes derived from adult rat atria. Atrial cardiocytes attached to microcarrier beads were maintained for up to 15 days under continuous superfusion in minichromatographic columns. The cultures were characterized for their ability to store, process, and release ANF and by immunofluorescence microscopy for ANF, desmin, and myosin. Nuclear staining using the fluorescent DNA stain Hoechst 33258 was carried out to determine the total number of cells in culture. Column eluates were assayed for ANF by radioimmunoassay and analyzed by reverse phase high-performance liquid chromatography. For comparison purposes, superfusion experiments using freshly isolated cardiocytes supported in Bio-Gel P2 were carried out. Freshly isolated atrial cardiocytes stored high molecular weight ANF (5.2 +/- 1.9 pmol/micrograms DNA) and released mostly (83.3 +/- 6.7%) low molecular weight ANF, at an average rate of 97 +/- 18 fmol.min-1 x micrograms-1 DNA. The cell content and the rate of release of ANF after 15 days in culture were 1.3 +/- 0.4 pmol/micrograms DNA and 1.7 +/- 0.4 fmol.min-1 x micrograms-1 DNA, respectively, and 62.7 +/- 6.3% of the released peptide was of a low molecular weight. There was no correlation between changes in cell population and the extent of processing. Cultures of noncardiocytes, superfused with exogenous proANF, did not significantly process proANF to a lower molecular weight peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

The stability of maize nuclei to restriction enzyme digestion differs with transcriptional activity

SUMMARYThe stability to restriction of nuclei obtained from plant cells of different transcriptional activity (dry embryo, root tip meristem and epicotyl protoplasts) has been studied. The morphological data obtained following fluorescent staining of nucleoid DNA with acridine orange indicate that the dry embryo structures are less stable than those actively involved in transcription, probably reflecting the lack of internal nuclear matrix structures in the dry embryo. The data support the suggestion for the key role these structures play in the functioning of the genome.

The influence of hydralazine on the vasculature, blood perfusion and chemosensitivity of MAC tumours

We have studied the influence of the peripheral vasodilator hydralazine (HDZ) on the vasculature and blood perfusion of two members of a series of subcutaneous murine adenocarcinomata of the colon (MAC tumours), and the influence of HDZ on the efficacy and/or toxicity of TCNU and melphalan. The fluorescent DNA stain Hoechst 33342, showed that HDZ caused a shutdown of tumour vasculature, related in magnitude to both dose and tumour differentiation state; 10 mg kg-1 caused an 80% vascular shutdown of well differentiated MAC 26 tumours, but only a 50% shutdown of the poorly differentiated MAC 15A tumours. 2.5 mg kg-1 was ineffective. The blood perfusion marker 99mTc-HMPAO showed that the normal perfusion of MAC tumours was consistently markedly less than that of lung, liver or kidneys (4-5% of lung perfusion). HDZ (10 mg kg-1) decreased MAC 26 perfusion by 63%, and that of MAC 15A by 20%. Again, 2.5 mg kg-1) was ineffective. Use of in vivo to in vitro clonogenic assays showed that HDZ (10 mg kg-1) potentiated the efficacy of melphalan (1-10 mg kg-1 i.p.) by a factor of 2.1, and increased the efficacy of TCNU (1-10 mg kg-1 i.v., factor = 1.7) when given 10 or 15 min respectively after dosing. However, the addition of HDZ increased the acute bone marrow toxicity of melphalan, but not that of TCNU. The clinical relevance of these results is discussed.

Microfluorimetric analysis of spatial and temporal patterns of immobilized cell growth

Pronounced spatial nonuniformities in cell density, physiology, and activity frequently arise within densely packed immobilized cell supports. For a more fundamental understanding of immobilized cell phenomena, we have developed high-resolution microfluorimetric procedures to analyze local variations in both immobilized cell loading and growth rate. Fluorescent staining of total cellular DNA provides a measure of local biomass density. Actively growing (DNA synthesizing) cells are marked by pulse-labeling newly synthesized DNA with the thymine analog, bromouracil. An immunofluorescent technique allows subsequent detection of spatial variations in DNA synthesis rates. These procedures enable the influence of mass-transfer limitations and other immobilization effects on cell distribution and activity to be readily quantified. We demonstrate this approach through analysis of the patterns of growth of Escherichia coli entrapped within Sr-alginate gel beads. The experimental techniques are potentially applicable to a variety of other aggregate cell systems.

Morpho-Functional Study of Vascular Fluorochrome Delivery to Lung and Liver Metastases of Lewis Lung Carcinoma (3Ll)

The growth of 3LL liver and lung metastases related to its vascular organization was studied by morphological and functional methods, using the Hoechst 33342 fluorescent DNA staining technique. Experimental liver and lung metastases were produced in syngeneic C57BL/6 mice by injection of 3LL tumor cells into a lateral tail vein or into the spleen, respectively. The resulting neoplasms were composed of large cells arranged in sheets with a thin irregularly distributed stroma. Scattered blood vessels with an open or closed lumen were observed within the tumor. Functional study of H33342 diffusion showed a single and reticular fluorescent pattern in liver metastases. In contrast, in lung metastases the fluorochrome diffusion revealed two different fluorescent patterns related to the location of the metastasis. Thus, parenchymal and subpleural metastasis presented a fluorescent pattern similar to that observed in the liver whereas metastases located around blood vessels and conducting airways never displayed fluorescence. In summary, our results suggest that the target metastatic organ and/or intra-organ location modulates the characteristics of metabolic exchange of the tumor cells in relation to the vascular organization of the metastatic focus.

Mycoplasma and Epidemic Group A Meningococcal Meningitis-Reply

In Reply. — As Drs Kenny and Foy point out, mycoplasma contamination of cell cultures is a common and insidious problem. We are confident that the M hominis isolated in our study came from the specimens and not the cell cultures due to the precautions taken to avoid and detect contamination. Ten uninoculated control cell cultures of each cell type were passaged in parallel with each batch of inoculated cultures; none of these controls became positive. As part of our quality assurance policies, each cell line is also tested monthly for mycoplasma at the Centers for Disease Control using an adenosine phosphorylase test (Mycotect, Gibco) and at the American Type Culture Collection (Rockville, Md) using both direct culture on Hayflick's medium and a DNA fluorescent staining test. 1,2 All three tests were consistently negative throughout the study period. Furthermore, when study specimens were positive for mycoplasma, all four cell types

Spatial Characterization of Glutathione Depletion in the KHT Sarcoma Using Flow Cytometry

Intravenous administration of the fluorescent DNA stain Hoechst 33342 to tumour-bearing mice was used to label cells proportionally to their proximity from the vasculature. Flow cytometry was used to sort cells from the tumour into populations based on their Hoechst 33342-derived fluorescence. The cell populations were then assayed for their glutathione (GSH) content and their radiosensitivity. Tumours from mice pretreated with buthionine sulphoximine (BSO) were compared with untreated animals. The major findings of this study suggest that the cellular GSH concentration within tumours decreases with distance from the vasculature, and that the GSH concentration within cells from all locations in the tumour can be depleted by enzymatically inhibiting its synthesis using BSO. This depletion of GSH resulted in a small degree of hypoxic radiosensitization of cells both distal and proximal to the vasculature.

Minor-groove recognition of the self-complementary duplex d(CGCGAATTCGCG)2 by Hoechst 33258: a high-field NMR study

The interaction of Hoechst 33258, a fluorescent DNA stain, has been studied by using the synthetic, self-complementary oligonucleotide duplex d(CGCGAATTCGCG)2. Spectrofluorometric Scatchard analysis indicated that there was only a single class of binding site and that the 1:1 complex had a dissociation constant of (3.47 +/- 0.1) X 10(-6) M at 25 degrees C. Spectroscopic titration by high-field 1H NMR confirmed the 1:1 complex and by means of ID and 2D (NOESY, COSY) techniques the binding site was defined as the minor groove formed by the AATT stretch. Plentiful cross-peaks were measurable and resonance doubling occurred because of the lifting of the diad symmetry of the oligonucleotide on ligand binding. Many individual resonances of both strands of the DNA could be assigned for the complex because of these features, along with the occurrence of slow exchange on the NMR time scale. The results of this NMR spectroscopic solution study were compared with those of previous X-ray crystallographic studies of the same complex. From nuclear Overhauser effect data measured for the complex, a detailed three-dimensional model was constructed with the aid of molecular graphics.

Flow‐Cytometric Method for Counting Very Low Numbers of Leukocytes in Platelet Products1

We provide a detailed description of a new method for counting very low numbers of leukocytes present in platelet products. The method uses fluorescent staining of leukocyte DNA using propidium iodide and flow cytometry. The assay is accurate over a range of leukocyte concentrations from 0.1 to 10 WBC/microliter. We validated the method using specially designed dilution studies and applied it to the measurement of leukocyte content in leukocyte-depleted platelet products prepared by apheresis or filtration. This method should prove useful in studies which address the clinical impact of leukocyte-depleted platelet products.

Intermittent blood flow in the KHT sarcoma--flow cytometry studies using Hoechst 33342

The administration of the fluorescent DNA stain, Hoechst 33342, to mice bearing the KHT sarcoma, combined with flow cytometry, can be used to select cells according to their proximity to functional vasculature. Different protocols of administration of Hoechst 33342 were used in order to differentiate between the presence of temporary and chronically hypoxic cells. The results show a large difference in radiosensitivity between cells close to, and distant from, functional vasculature. However, this pattern of radiosensitivity is observed only when the staining period with Hoechst 33342 is short and coincides with the period of irradiation. When the radiation treatment is temporally divorced from the staining period then the radiosensitivity and staining intensity are not related. This result can be interpreted as indicating that hypoxic cells exist within this tumour as a result of fluctuations in tumour blood flow.

Probing DNA structure and function with a multiwavelength fluorescence confocal laser microscope

Three levels of organization in DNA structure in the interphase cell nucleus are assessed by confocal laser scanning microscopy: (i) the conformational state of the double helix; (ii) the distribution of eu- and heterochromatin; and (iii) the localization of replication complexes throughout S phase. Multi-parameter measurements were carried out in each optical section using two laser sources and combined stereoscopic reconstructions were used to assess the co-localization of nuclear components. DNA is highly polymorphic and can adopt a variety of different helical conformations as well as unusual structures (curved, cruciform, multi-stranded). We have assessed by laser scanning microscopy the presence of left-handed Z-DNA in polytene chromosomes of Diptera as well as the spatio-temporal distribution of Z-DNA binding proteins in whole-mount Drosophila embryos and ovaries. We have determined the 3-D distribution of replication sites relative to heterochromatin regions, nucleoli and nuclear membrane by using short pulses of BrdU incorporation in synchronized mouse and human fibroblasts. Replication sites were visualized with a monoclonal anti-BrdU antibody combined with DNA fluorescent staining and antibody labelling of nuclear lamin. The implications of dynamic DNA movement and structural rearrangement to the organization of the nucleus in domains are discussed.

Survey of mycoplasma infections in cell cultures and a comparison of detection methods

A total of 1424 cell cultures was assayed for mycoplasmas by microbiological culture and fluorescent DNA staining. Of these cultures, 412 (29%) were infected with mycoplasmas. The most frequently occurring mycoplasma species were Mycoplasma orale (34%), M. hyorhinis (26%), M. arginini (21%), M. fermentans (13%) and Acholeplasma laidlawii (5%). A few isolates each of M. hominis, M. pulmonis and M. bovis were also detected. When detection methods were compared, microbiological culture produced false-negative results for 0.7% (3 of 412) of the infected cell cultures. DNA staining performed directly on the cells was falsely negative in 2.4% (5/207) of the mycoplasma-infected cultures that were compared, DNA staining performed on indicator cells was falsely negative in 3.1% (7/226). False positives appeared in direct DNA-staining in 1.8% (7/386) of the mycoplasma-free cultures and with DNA staining on indicator cells in 0.5% (3/620). For 11% of the cell cultures, the reading of the DNA staining was ambiguous. With DNA staining on indicator cells, 10% of the test results were ambiguous, but by further passage and staining on new indicator cells it was possible to get a definite diagnosis.

Minicell-forming mutants of Escherichia coli: production of minicells and anucleate rods.

The Escherichia coli minB mutant originally isolated is known to septate at cell poles to form spherical anucleate minicells. Three new minicell-producing mutants were isolated during a screening by autoradiography for chromosome partition mutants giving rise spontaneously to normal-sized anucleate cells. These min mutants were affected close to or in the minB locus. Autoradiography analysis as well as fluorescent staining of DNA showed that in addition to minicells, these strains and the original minB mutant also spontaneously produced anucleate rods of normal size and had an abnormal DNA distribution in filaments. These aberrations were not associated with spontaneous induction of the SOS response. Inhibition of DNA synthesis in these mutants gave rise to anucleate cells whose size was longer than unit cell length, suggesting that the min defect allows septation to take place at normally forbidden sites not only at cell poles but also far from poles. Abnormal DNA distribution and production of anucleate rods suggest that the Min product(s) could be involved in DNA distribution.

Ploidy, proliferative activity, cluster differentiation antigen expression and clinical remission in high-grade non-Hodgkin's lymphoma.

Using a large range of monoclonal antibodies to specific cluster differentiation antigens the phenotypes of a series of high-grade non-Hodgkin's lymphomas of B- and T-cell type were investigated. Cell ploidy and proliferative fraction were assessed by fluorescent staining of DNA and flow cytometry and data on the incidence of complete clinical remission were obtained. With the exception of some lymphoblastic lymphomas, high-grade B-cell lymphomas normally expressed the pan B-cell antigens CD19 and CD22 but only immunoblastic lymphomas consistently expressed the pan B marker CD20. Variable, generally weak expression of CD21 was observed whilst CD23 expression was most prevalent in rapidly proliferative cases and in Burkitt's and centroblastic lymphomas. A rapidly proliferative, multilobated B-cell lymphoma displayed phenotypic properties intermediate between centroblastic and immunoblastic lymphomas. The T-cell lymphomas generally showed low proliferative activity and expression of CD4 prevailed over CD8. Most cases also showed CD2 and CD5 positivity with some also showing CD3 and CD7 expression. Patients with rapidly proliferative diploid or DNA aneuploid tumours obtained complete remission more readily than patients with lowly proliferative diploid tumours. An excess of early deaths occurred among T-cell cases.

A Combined Immunofluorescence-DNA-Fluorescence Staining Technique for Enumeration of Thiobacillus ferrooxidans in a Population of Acidophilic Bacteria

An antiserum raised against whole cells of Thiobacillus ferrooxidans was allowed to react with a variety of acidophilic and nonacidophilic bacteria in an enzyme-linked immunosorbent assay and an indirect immunofluorescence assay. Both experiments demonstrated that the antiserum was specific at the species level. This preparation was used to evaluate the role of T. ferrooxidans in the microbial desulfurization process. Leaching experiments were performed, and the numbers of T. ferrooxidans cells and other bacteria were estimated by using a combined immunofluorescence-DNA-fluorescence staining technique that was adapted for this purpose. Nonsterile coal samples inoculated with T. ferrooxidans yielded high concentrations of soluble iron after 16 days. After this period, however, T. ferrooxidans cells could no longer be detected by the immunofluorescence assay, whereas the DNA-fluorescence staining procedure demonstrated a large number of microorganisms on the coal particles. These results indicate that T. ferrooxidans is removed by competition with different acidophilic microorganisms that were originally present on the coal.