Flow cytometric analysis of proliferation associated nuclear antigens using washless staining of unfixed cells

Abstract

The expression of different proliferation associated nuclear antigens was analyzed using a washless double-staining method and flow cytometry. It is a simple and rapid two-step procedure which can be performed on low cell numbers. A series of hematopoietic cell lines and fresh lymphoma cells were tested and the methodology was found to be applicable to a number of nuclear antigens (PCNA, Ki-67, p105, MPM-2, fibrillarin). For PCNA, the detectability was dependent on the type of antibody used. The immunofluorescence pattern observed by microscopy was altered for antigens stained by the washless technique in comparison with the pattern obtained with fixed cells. With the washless method, detailed cell cycle analysis could be obtained by dual parameter analysis of PCNA and Ki-67.

Three parameter flow cytometric analysis for simultaneous detection of cytokeratin, proliferation associated antigens and DNA content

“…In this study we have presented a three-color staining protocol that is based on the fixation and staining procedure for bivariate PCNA/DNA staining as originally described by Landberg and Roos (13). This protocol allows detailed cell cycle analysis of epithelial cells in heterogeneous mixtures of infiltrating normal cells and tumor cells.…”

Identification of S-phase cells with PC10 antibody to proliferating cell nuclear antigen (PCNA) by flow cytometric analysis.

“…PCNA is expressed in nuclei of proliferating cells (4-7,11,15,18,20,27,32-36) and has been advocated for assessment of biological behavioral measurement in many fields of research (1,2,16,24,29). Nevertheless, it has been commonly reported that PCNA has been detected in S-phase cells when an autoantibody in sera from patients with SLE is used (19,21,22,26). The discrepancy in PCNA reactivity can be explained by differences between epitopes that the auto-and monoclonal antibodies recognize (13).…”

Flow Cytometric Evaluation of Proliferating Cell Nuclear Antigen Expression in Human Hematopoietic Malignancies

“…One detergent extractable PCNA form is present in increased amounts in proliferating cells as compared with resting cells. A second form of PCNA is detergent resistant and associated with the DNA replication complex (16). The PCNA protein can be detected by monoclonal antibodies using immunohistochemical techniques or by flow cytometry (FCM) (8,16).…”

“…A second form of PCNA is detergent resistant and associated with the DNA replication complex (16). The PCNA protein can be detected by monoclonal antibodies using immunohistochemical techniques or by flow cytometry (FCM) (8,16). Depending on the cell preparation technique used for FCM analysis, selective detection of PCNA in S-phase cells (PCNA-S) or PCNA in proliferating cells (PCNA-tot) can be achieved (17).…”