Rapid hypotonic method for flow cytofluorometry of monolayer cell cultures. Some pitfalls in staining and data analysis.

Fried, Jerrold; Perez, Almudena Chaves; Clarkson, Bayard D.

doi:10.1177/26.11.82573

Cited by 122 publications

(50 citation statements)

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“…A number of oligonucleotides served as negative controls in these experiments, including sense, reverse and mutated bcl-2 (Smith et al, 1995), antisense and mutated J H , and mutated c m . Effects secondary to sequence motifs, such as polyguanosine (reviewed in Cleary et al, 1986;Freid et al, 1978), and to methylation of CpG (Krieg et al, 1995) have also been described. Our active oligonucleotides have no polyguanosine stretches, while both Values expressed as mean^SD of duplicate values from two separate experiments.…”

Section: Discussionmentioning

confidence: 99%

“…While antisense oligonucleotides are designed to be sequence-specific regulators of gene expression, sequenceindependent effects, perhaps because of their polyanionic characteristics, have been observed (reviewed in Freid et al, 1978;Cleary et al, 1986). A number of oligonucleotides served as negative controls in these experiments, including sense, reverse and mutated bcl-2 (Smith et al, 1995), antisense and mutated J H , and mutated c m .…”

Section: Discussionmentioning

confidence: 99%

“…For analysis of DNA content per cell, cells were treated with the indicated oligonucleotide (80 mg/ml) or 1 mmol/l VP-16 for 48 h. Cells were lysed in hypotonic solution containing 50 mg/ml propidium iodide (Freid et al, 1978) and analysed (FACScan, Becton-Dickinson). As a more specific marker of apoptosis, cells were stained with phycoerythrin (PE)-Cy5-labelled Apo2.7 (Immunotech, Marseille, France) or PE-IgG control (Koester et al, 1997).…”

Section: Methodsmentioning

confidence: 99%

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Antisense oligonucleotides complementary to immunoglobulin sequences of BCL‐2/immunoglobulin fusion transcript induce apoptosis of t(14;18) lymphoma cells

et al. 2000

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Summary. Antisense oligodeoxyribonucleotides directed at the bcl-2 translational start site downregulate bcl-2 and inhibit growth of the t(14;18)-positive lymphoma line WSU-FSCCL. Non-specific downregulation of bcl-2 expression is expected to be toxic to normal cells as well. The t(14;18) translocation results in a fusion transcript containing the entire bcl-2 coding sequence with a 3 H breakpoint fused to the immunoglobulin J H region and the c m heavy chain. We postulated that these immunoglobulin sequences would be a specific target for downregulation of the fusion gene. Here, we have demonstrated that antisense oligodeoxyribonucleotides targeted to immunoglobulin c m sequences downregulate bcl-2 protein expression and induce apoptosis of WSU-FSCCL cells. Inhibiting growth of malignant cells by targeting non-oncogenic sequences other than breakpoints of fusion transcripts expands the potential for tumourspecific genetic manipulation.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Antisense oligonucleotides complementary to immunoglobulin sequences of BCL‐2/immunoglobulin fusion transcript induce apoptosis of t(14;18) lymphoma cells

et al. 2000

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“…Cells were prepared for flow cytometric analysis as described above (Fried et al, 1978;Davidoff & Mendelow, 1993a, b). 1 ml propidium iodide (PI) cocktail (1 g/l Triton X100; 1 g/l sodium citrate; 50 mg/l PI in phosphate-buffered saline, pH 7·3) was added to the tubes containing 1 × 10 6 cells.…”

Section: Patients Materials and Methodsmentioning

confidence: 99%

Evaluation of DNA analysis for evidence of apoptosis in megaloblastic anaemia

et al. 1997

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Summary. This study involved DNA analysis of bone marrow cells of 15 patients with megaloblastic anaemia. The diagnosis was based on the morphological changes seen in the bone marrow, associated with either a low red cell folate or serum vitamin B12 level and an adequate response to appropriate therapy as confirmation of the diagnosis. Flow cytometric DNA analysis showed an increase in the S and G 2 phases of the cell cycle, but conventional agarose gel electrophoretic DNA analysis did not confirm the characteristic 'ladder pattern' which might have been expected in classic apoptosis.In addition, cells showing morphological changes suggestive of apoptosis, such as nuclear condensation and fragmentation, did not show evidence of DNA fragmentation using the ApopTag TM in situ digoxigenin nucleotide labelled, peroxidase detection system. Further studies using annexin V flow cytometric analysis and pulsed field gel electrophoresis were also unable to detect evidence of apoptosis as a significant cause of cell death in megaloblastic anaemia.

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“…At 24, 48 and 72 h post-infection, the cells were detached, stained with trypan blue and the number of viable cells was counted with a Neubauer chamber. The same conditions were used to evaluate the cell cycle stage via flow cytometry, as described by Fried et al (1978).…”

Section: Cells and Viruses -The Aedes Albopictus Cell Linementioning

confidence: 99%

Genetic and biological characterization of a densovirus isolate that affects dengue virus infection

Mosimann

Bordignon

Mazzarotto

et al. 2011

Mem. Inst. Oswaldo Cruz

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Brevidensoviruses have an encapsidated, single-stranded DNA genome that predominantly has a negative polarity. In recent years, they have received particular attention due to their potential role in the biological control of pathogenic arboviruses and to their unnoticed presence in cell cultures as contaminants. In addition, brevidensoviruses may also be useful as viral vectors. This study describes the first genetic and biological characterization of a mosquito densovirus that was isolated in Brazil; moreover, we examined the phylogenetic relationship between this isolate and the other brevidensoviruses. We further demonstrate that this densovirus has the potential to be used to biologically control dengue virus (DENV) infection with in vitro co-infection experiments. The present study provides evidence that this densovirus isolate is a fast-spreading virus that affects cell growth and DENV infection

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Rapid hypotonic method for flow cytofluorometry of monolayer cell cultures. Some pitfalls in staining and data analysis.

Cited by 122 publications

References 0 publications

Antisense oligonucleotides complementary to immunoglobulin sequences of BCL‐2/immunoglobulin fusion transcript induce apoptosis of t(14;18) lymphoma cells

Antisense oligonucleotides complementary to immunoglobulin sequences of BCL‐2/immunoglobulin fusion transcript induce apoptosis of t(14;18) lymphoma cells

Evaluation of DNA analysis for evidence of apoptosis in megaloblastic anaemia

Genetic and biological characterization of a densovirus isolate that affects dengue virus infection

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