Determination of the growth fraction in cell suspensions by flow cytometry using the monoclonal antibody Ki-67

Schwarting, Roland; Gerdes, Johannes; Niehus, J.; Jaeschke, Lothar; Stein, Harald

doi:10.1016/0022-1759(86)90384-4

Cited by 137 publications

(80 citation statements)

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“…To address this issue, we assessed the distribution of actively cycling cells in circumvallate and fungiform taste papillae, with the expectation that a few stem cells and all transit amplifying cells would be mitotically active. We employed 4 immunomarkers to identify dividing cells: 1) proliferating cell nuclear antigen (PCNA) present in all phases of cell cycle except early G1 [54-56]; 2) Ki-67, which detects all phases in actively cycling cells, i.e., G1, S, G2, and M [57]; 3) BrdU, which is incorporated into DNA during S phase [58]; and 4) Phospho-Histone 3 (pH3) which marks cells in M phase (reviewed by Norbury and Nurse, 1992) [59]. The vast majority of basal epithelial cells of both the fungiform and circumvallate taste papillae were actively cycling, as evidenced by broad Ki-67 (Figure 6) and PCNA-IR (Figure 7 - red), while a small percentage of cells were mitosing and were pH3-IR (Figure 7 - green).…”

Section: Resultsmentioning

confidence: 99%

“…If so, these cells should express markers of actively cycling cells, including Ki-67 and BrdU incorporation during S phase DNA synthesis. However, double labeling with Ki-67, which labels mitotically active cells in all phases of the cell cycle [57], showed that these BMP4-ß-gal expressing cells were not actively cycling. We further confirmed this lack of mitotic activity with additional markers of cell proliferation (PCNA and pH3), and in short duration birthdating studies employing BrdU.…”

Section: Discussionmentioning

confidence: 99%

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Differential expression of a BMP4 reporter allele in anterior fungiform versus posterior circumvallate taste buds of mice

Nguyen

Barlow

2010

BMC Neurosci

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BackgroundBone Morphogenetic Protein 4 (BMP4) is a diffusible factor which regulates embryonic taste organ development. However, the role of BMP4 in taste buds of adult mice is unknown. We utilized transgenic mice with LacZ under the control of the BMP4 promoter to reveal the expression of BMP4 in the tongues of adult mice. Further we evaluate the pattern of BMP4 expression with that of markers of specific taste bud cell types and cell proliferation to define and compare the cell populations expressing BMP4 in anterior (fungiform papillae) and posterior (circumvallate papilla) tongue.ResultsBMP4 is expressed in adult fungiform and circumvallate papillae, i.e., lingual structures composed of non-taste epithelium and taste buds. Unexpectedly, we find both differences and similarities with respect to expression of BMP4-driven ß-galactosidase. In circumvallate papillae, many fusiform cells within taste buds are BMP4-ß-gal positive. Further, a low percentage of BMP4-expressing cells within circumvallate taste buds is immunopositive for markers of each of the three differentiated taste cell types (I, II and III). BMP4-positive intragemmal cells also expressed a putative marker of immature taste cells, Sox2, and consistent with this finding, intragemmal cells expressed BMP4-ß-gal within 24 hours after their final mitosis, as determined by BrdU birthdating. By contrast, in fungiform papillae, BMP4-ß-gal positive cells are never encountered within taste buds. However, in both circumvallate and fungiform papillae, BMP4-ß-gal expressing cells are located in the perigemmal region, comprising basal and edge epithelial cells adjacent to taste buds proper. This region houses the proliferative cell population that gives rise to adult taste cells. However, perigemmal BMP4-ß-gal cells appear mitotically silent in both fungiform and circumvallate taste papillae, as we do not find evidence of their active proliferation using cell cycle immunomarkers and BrdU birthdating.ConclusionOur data suggest that intragemmal BMP4-ß-gal cells in circumvallate papillae are immature taste cells which eventually differentiate into each of the 3 taste cell types, whereas perigemmal BMP4-ß-gal cells in both circumvallate and fungiform papillae may be slow cycling stem cells, or belong to the stem cell niche to regulate taste cell renewal from the proliferative cell population.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Differential expression of a BMP4 reporter allele in anterior fungiform versus posterior circumvallate taste buds of mice

Nguyen

Barlow

2010

BMC Neurosci

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“…We have chosen to investigate the effects of increased salt concentration on the detectability of three antigens that are well characterized with respect to their molecular structure, appear to be promising as tumor prognostic markers in oncology, and to which the antibodies are commercially available. Specifically, we studied the Proliferation Cell Nuclear Antigen (PCNA); (5, 16,18), the nucleolar antigen p120 (11,12), and the protein detected by Ki-67 antibody (1, 13,21). The data show that, indeed, a significant increase in the binding of the antibodies directed toward PCNA and Ki-67 antigen occurs when cells are fixed at increasing salt concentrations.…”

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confidence: 99%

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

1992

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This study was aimed at revealing whether or not ionic interactions between the epitope of the antigen detected by Ki-67 antibody, or the proliferation-associated proteins PCNA or p120, and neighboring cellular constituents impede detectability of these antigens in HL-60 cells by indirect immunofluorescence assay. To this end, the ionic strength (NaCl concentration) of the solutions in which cells were suspended during their fixation with 0.5% paraformaldehyde was increased, to up to 1.65 M NaCl, to weaken the intra-and/or intermolecular ionic interactions during the process of crosslinking, and the cells were then immunostained. Fluorescence of cells reacting with Ki-67 antibody was maximally increased after their treatment with 1.15 M NaCI; the average increase was nearly 110% above the level seen with the standard methodology utilizing 0.15 M NaCl. The increase was greater for cells in the G, phase of the cell cycle compared to cells in S or G,. Fluorescence of cells stained with the PCNA antibody was maximally enhanced after cell treatment with 0.65 M NaCl. The enhancement, however, varied depending on the source of the antibody; it was nearly 200% in the case of the antibody provided by Boehringer and over 100% by DAKO. Detection of the nucleolar antigen p120 was not significantly affected by 0.65-1.65 M NaCl. The data indicate that ionic interactions between cellular constituents indeed play a role in masking the epitope of PCNA and the antigen detected by Ki-67. These results also suggest that to enhance the detectability of the intracellular proteins, the current immunocytochemical methods should be modified and customized for each particular antigen in terms of selection of the optimal ionic strength during the fixation procedure. o 1992 Wiley-Liss, Inc.

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“…The "The various staining methods were all tested on each of two PHA stimulation culture series from the same donor. Parallel cultures were continuously labeled with 20 and 50 p. M BrdUrd from 24 h after PHA and were both included in the data. In methods VI-VIII, a threshold in green fluorescence was set for optimal discrimination of BrdUrd nonincubated lymphocytes as a BrdUrd-negative population.…”

Section: Resultsmentioning

confidence: 99%

“…Methods for combined analysis of surface markers and Ki-67 antigen have been reported by Schwarting et al (20) using acetone-fixed cells and by Drach et al (6) using fixation with periodatellysineiparaformaldehyde at -10°C. Methods for combined analysis of DNA and Ki-67 antigen have been reported by Baisch and Gerdes (1,2) and Sasaki et al (19) using acetone fixed cells, by Landberg et al (12) using ethanol fixed cells, and by Palutke et al (16) using nuclear suspensions prepared with formaldehyde, detergent, ethanol, and citrate.…”

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confidence: 99%

Washless double staining of unfixed nuclei for flow cytometric analysis of DNA and a nuclear antigen (Ki‐67 or bromodeoxyuridine)

et al. 1991

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Washless methods for double staining of nuclear antigen and DNA in unfixed nuclei were compared with established methods for staining of fixed cells. The methods were tested on phytohemagglutinin (PHA)-stimulated normal human blood lymphocytes for the double staining of 1) Ki-67 antigen and DNA and 2) bromodeoxyuridine (BrdUrd) and DNA, in continuously BrdUrd-labeled cells. With respect to the discrimination between antigen-positive and -negative subpopulations, there was no statistically significant differences between the results from direct (Ki-67) or indirect (Ki-67 or BrdUrd) washless staining of unfixed nuclei and the results from staining of fixed cells. Washless staining of unfixed nuclei was found to be rapid and simple and resulted in greater precision of the DNA analysis and in less aggregation and loss of cells.Key terms: Ki-67 antibody, anti-bromodeoxyuridine antibody, DNA synthesis, cell cycle, cell proliferation, lymphocyte activation, HL-60 cellsThe analysis of cell cycle distributions by flow cytometric DNA measurement (static parameter) may be extended with a cell kinetic (dynamic) parameter using simultaneous measurement of bromodeoxyuridine (BrdUrd). BrdUrd is incorporated into DNA-synthesizing cells and can be stained by use of anti-BrdUrd antibody, which recognizes BrdUrd, but only in singlestranded DNA (5,101. The Ki-67 antibody recognizes a nuclear antigen naturally expressed in cycling cells and may be more practical for clinical investigations than the anti-BrdUrd antibody (2,7,8). This is because its use is not dependent on complicated vital or supravital labeling and subsequent denaturation of the labeled molecule.For flow cytometric application of the Ki-67 antibody, several methods have been described, all using fixed cells and a large number of washings. Methods for combined analysis of surface markers and Ki-67 anti- In this study, we apply new methods for washless double staining of unfixed nuclear suspensions (13) to achieve a flow cytometric analysis of the correlation between the nuclear contents of DNA and a specific antigen. The feasibility of this methodological principle is demonstrated for two proliferation-associated nuclear antigens, by combined measurements in phytohemagglutinin (PHA)-stimulated lymphocytes of 1) Ki-67 antigen and DNA and 2) continuously incorporated BrdUrd and DNA. Furthermore, the washless methods for staining of unfixed nuclei are compared with established methods for staining of fixed cells (1,51.

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Determination of the growth fraction in cell suspensions by flow cytometry using the monoclonal antibody Ki-67

Cited by 137 publications

References 7 publications

Differential expression of a BMP4 reporter allele in anterior fungiform versus posterior circumvallate taste buds of mice

Differential expression of a BMP4 reporter allele in anterior fungiform versus posterior circumvallate taste buds of mice

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

Washless double staining of unfixed nuclei for flow cytometric analysis of DNA and a nuclear antigen (Ki‐67 or bromodeoxyuridine)

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