Effect of ionic strength in immunocytochemical detection of the proliferation associated nuclear antigens p120, PCNA, and the protein reacting with Ki‐67 antibody

Bruno, Silvia; Gorczyca, Wojciech; Darzynkiewicz, Zbigniew

doi:10.1002/cyto.990130508

Cited by 32 publications

(21 citation statements)

References 18 publications

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“…It has been found that the staining of some nuclear antigens is improved following fixation under high ionic strength (23), or following treatment with alcohols, but we did not observe that with any of the histone marks tested. However, further developmental work could be done in this area, for example, testing alternative fixatives or fixation conditions.…”

Section: Discussioncontrasting

confidence: 99%

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The study of epigenetic mechanisms based on the analysis of histone modification patterns by flow cytoametry

et al. 2013

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Epigenetic regulation of genes involved in cell growth, survival, or differentiation through histone modifications is an important determinant of cancer development and outcome. The basic science of epigenetics uses analytical tools that, although powerful, are not well suited to the analysis of heterogeneous cell populations found in human cancers, or for monitoring the effects of drugs designed to modulate epigenetic mechanisms in patients. To address this, we selected three clinically relevant histone marks (H3K27me3, H3K9ac, and H3K9me2), modulated their expression levels by in vitro treatments to generate high and low expressing control cells, and tested the relative sensitivity of candidate antibodies to detect the differences in expression levels by flow cytometry using a range of sample preparation techniques. We identified monoclonal antibodies to all three histone marks that were suitable for flow cytometry. Staining intensities were reduced with increasing formaldehyde concentration, and were not affected by ionic strength or by alcohol treatment. A protocol suitable for clinical samples was then developed, to allow combined labeling of histone marks and surface antigens while preserving light scatter signals. This was applied to normal donor blood, and to samples obtained from 25 patients with leukemia (predominantly acute myeloid leukemia). Significant cellular heterogeneity in H3K9ac and H3K27me3 staining was seen in normal peripheral blood, but the patterns were very similar between individual donors. In contrast, H3K27me3 in particular showed considerable inter-patient heterogeneity in the leukemia cell populations. Although further refinements are likely needed to fully optimize sample staining protocols, "flow epigenetics" appears to be technically feasible, and to have potential both in basic research, and in clinical application. V C 2013 International Society for Advancement of Cytometry Key terms epigenetics; histone; leukemia; flow cytometry; drug treatment WITH the rapid development and application of sequencing technologies, it is becoming evident that alterations at the genome level are unlikely to explain the heterogeneity of human cancers (1), and there is increasing recognition that epigenetic mechanisms also play a major role in cancer development and progression. The epigenetic regulation of genes is complex, and occurs at the levels of DNA, histone proteins, and RNA (2-4). DNA methylation at promoter regions can cause long term gene silencing, whereas histone modifications, predominantly affecting lysines in the N-terminal tails of the core histone proteins H3 and H4, are more dynamic and associated with relatively short term plasticity.The most frequent lysine modifications of histone proteins ("marks") involve acetylation and methylation, although phosphorylation, ubiquitylation and sumoylation can also occur at these sites. Histone acetylation, which alters the charge distribution, is typically associated with open chromatin that enables gene transcription, whereas meth...

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Section: Discussioncontrasting

confidence: 99%

“…Original Article the staining of some nuclear antigens (23). OCI-AML2 cells were fixed with 4% formaldehyde in concentrations of NaCl ranging from 0 to 1.5 M, but we did not observe any significant effect on staining intensity (data not shown).…”

Section: Optimization Of Flow Cytometry Protocols For Tissue Culturementioning

confidence: 73%

The study of epigenetic mechanisms based on the analysis of histone modification patterns by flow cytoametry

et al. 2013

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“…In addition, immunodetec tion of endogenous proteins is limited to those species exhibiting cross-reactivity and proliferation antigens tend to be labile and have fastidious fixation requirements. A number of factors have been shown to influence PCNA immunoreactivity, including fixation time [29][30][31][32], fixa tive composition [33,34] and the source of antibody [33]. In addition, immunoreactive PCNA may be absent in cer tain proliferating neoplastic cells [35] and present in nor mal, nonproliferating tissues [36,37], In the current experiment, both fixation time and section thickness influenced PCNA immunoreactivity.…”

Section: Endogenous Proliferation Markersmentioning

confidence: 97%

Kinetics of Bromodeoxyuridine Uptake by Smooth Muscle Cells after Arterial Injury

London

Mayberg²

1994

J Vasc Res

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The time course of 5-bromo-2’-deoxyuridine (BrdU) uptake by medial and neointimal smooth muscle cells was examined in rat carotid arteries at periods of 1-20 days after balloon catheter injury. DNA-incorporated BrdU was determined by the indirect immunoperoxidase technique in both plastic- and paraffin-embedded specimens. The BrdU labeling index (LI) peaked at 48 h in the media (19.9% paraffin, 9.4% plastic) and at 5 days in the neointima (55.4% paraffin, 60.2% plastic). Immunohistochemistry for the endogenous marker proliferating cell nuclear antigen was variable and generally less reliable than BrdU. Using total viable medial smooth muscle cell number as an index of medial injury, the LI for BrdU was directly proportional to the extent of injury at 48 h, and correlated with loss of actin immunoreactivity. BrdU immunohistochemistry is a reliable alternative to [³H]-thymidine autoradiography for short-term labeling in this model. Proliferation of smooth muscle cells in this model is likely related to the degree of injury to the media.

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“…The coefficient of variation of Go/G 1 ceils regarding normal population ranged from 2.1 to 6.4 (mean 3.2), and the aneuploid population was from 1.7 to 7.3 (mean 3.2). Ki-67 expression was analyzed by Lysis II using a bivariate fluorescent distribution of cells stained by PI and fluorescein-labeled antibodies to Ki-67 in dot plots [5]. The FCM result of DNA ploidy was considered positive when aneuploidy was detected.…”

Section: Flow Cytometrymentioning

confidence: 99%

DNA ploidy, S-phase, and Ki-67 antigen expression in the evaluation of cell content of pleural effusions

Sikora¹,

Dworacki²,

Zeromski³

1996

Lung

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Cells from pleural effusions (28 malignant and 14 nonmalignant with reactive mesothelial cells) were examined in parallel by means of conventional cytology and multiparametric flow cytometry. The latter included the evaluation of DNA ploidy, the calculation of DNA index (DI), the determination of S phase fraction (SPF), and cell proliferation associated with Ki-67 antigen expression. Propidium iodide was used for the DNA staining, and fluorescein-conjugated monoclonal mouse antibodies were applied to the human Ki-67 antigen staining. The specimens were analyzed by the Becton Dickinson (BD) FACScan apparatus. 10(4) events were collected, and thereafter the obtained data were analyzed by the software Lysis II and CellFit (BD) for DNA and Ki-67 data analysis, respectively. Out of the 28 cases examined, 18 (64%) of the malignant effusions were aneuploid. All of the benign effusions were typically diploid. The average SPF of benign, malignant diploid, and malignant aneuploid cases was 6, 6, and 30 respectively. The comparison between SPF and DI in malignant cases showed a significant correlation (p < 0.001). The average of Ki-67-positive cells that were benign, malignant diploid, and malignant aneuploid were 11, 19, and 35, respectively. There was a significant correlation between Ki-67 expression, SPF (p < 0.05), and the percent of the aneuploid cell population (p = 0.01). The sensitivity of conventional cytology, the determination of DNA ploidy and of Ki-67 expression, was 80, 64, and 92%, respectively; specificity was 100% for cytology and DNA ploidy and 46% for Ki-67 expression. These data show that Ki-67 antigen expression is a potentially useful adjunct to cytometric DNA content analysis, determination of SPF, and conventional cytology in the diagnosis of malignant pleural effusions.

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Effect of ionic strength in immunocytochemical detection of the proliferation associated nuclear antigens p120, PCNA, and the protein reacting with Ki‐67 antibody

Cited by 32 publications

References 18 publications

The study of epigenetic mechanisms based on the analysis of histone modification patterns by flow cytoametry

The study of epigenetic mechanisms based on the analysis of histone modification patterns by flow cytoametry

Kinetics of Bromodeoxyuridine Uptake by Smooth Muscle Cells after Arterial Injury

DNA ploidy, S-phase, and Ki-67 antigen expression in the evaluation of cell content of pleural effusions

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