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

Abstract

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.

Absence of primary cilia in cell cycle‐arrested human breast cancer cells

“…Next, the orthotopic and metastatic xenograft tumor cells in mice that formed metastatic lesions (xenograft #4) were sorted by FACS, and the cell cycle of the cancer cells was analyzed by an anti‐Ki‐67 mAb and propidium iodide (PI) staining (Larsen et al . ) (Fig. C, F).…”

Improved single laser measurement of two cellular antigens and DNA-ploidy by the combined use of propidium iodide and TO-PRO-3 iodide

“…Also, cell-cycle-dependent antigen expression, e.g., S-phase specific PCNA (13), Ki-67 (14), and cyclins (9,10), can be studied more accurately when cells are also stained for DNA. Therefore, we conclude that the addition of TP3 eliminates the spectral cross talk problems caused by PI and improves the analysis of antigen expression in heterogeneous cell populations in combination with DNA-ploidy measurements.…”

Downregulation of CXCR4 in Metastasized Breast Cancer Cells and Implication in Their Dormancy

“…The cells of the dissociated PDX stained with Alexa Fluor 488-conjugated mouse anti-human HLA-A, B, C mAb and the cell line-derived tumor cells expressing ZsGreen1 were analyzed and sorted using FACS Aria I (BD Biosciences). In the cell cycle analysis, the sorted cells were subsequently permeabilized by a detergent solution (0.5% Triton-X 100 in PBS, 0.5 mM EDTA, pH 7.2), and then stained with propidium iodide (Sigma) with addition of RNase (Sigma) as previously described [21]. In the ex vivo culture experiment, the high expression level of cell surface CXCR4 was determined as the signal level that includes more than 50% of the orthotopic cancer cells and less than 5% of the metastasized cancer cells in the lung on average.…”