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A selective involvement of protein kinase C-zeta (PKC-zeta) in the events regulating cell proliferation has been recently proposed. Here we report a flow cytometric method allowing the simultaneous association of intracellular PKC-zeta expression or phosphorylation with each cell cycle phase. Current methods for flow cytometry analysis were applied to several cell lines and compared to the method developed in our laboratory. The latter includes 2% paraformaldehyde (PFA), as fixing agent, a permeabilization/saturation step by means of a solution containing 150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl pH 7.4, 0.05% NP-40, 0.25% lambda-carrageenan and 0.02% NaN3, followed by labelling with a primary antibody (PKC-zeta or P-PKC-zeta) and with the appropriate FITC-conjugated secondary antibody. Cells processed by such a method disclosed no substantial modification of light scattering features with respect to live cells. In addition, stainability with anti-PKC-zeta or anti-P-PKC-zeta antibodies was well preserved while stoichiometric staining of DNA with PI enabled accurate cell cycle analysis. Results show that a distinct upregulation of P-PKC-zeta in G2/M phase occurs. The method here described, therefore, represents a simple, reproducible and conservative assay for a simultaneous assessment of intracellular PKC or P-PKC modulations within each cell cycle phase. (c) 2006 Elsevier B.V. All rights reserved.

In this report, we have evaluated the effects of a TransFixR-based stabilisation technique on leukocyte scatter characteristics, immunophenotyping, membrane permeability, absolute cell counting and morphology to extend previously reported flow cytometric data focused on the lymphocyte population. We show that scatter characteristics, immunophenotyping and absolute cell counting are well preserved, particularly in the lymphocyte population. Nevertheless, a general increase in membrane permeability, evaluated by propidium iodide (PI) uptake, was observed in TransFixR-treated leukocyte subsets. Ultrastructural observations show selective morphological preservation (up to 10 days of storage) of lymphocytes and, to a lesser extent, of monocytes. In contrast, granulocytes have necrosis-like features, although the plasma membrane seems well preserved. Therefore, electron microscopy observations reflect modifications induced in different cell populations as evidenced by flow cytometry (FC). The data indicate that this short-term stabilisation method is particularly suitable for the analysis of human lymphocytes and it is a good procedure for quality control programmes for inter- and intra-laboratory performance evaluation; good results are obtained with respect to antigen definition and absolute cell counting procedures. Any apoptotic pathways in leukocyte subsets are blocked for at least 10 days.

The recognition of effector cell populations that are able to actively form conjugates with target cells is of major importance in studies of lymphocyte cytotoxicity. A number of methodologies have been described to identify the conjugates and count them, but there have been few studies of the birding capability of the different subsets of effector cells involved in the conjugation phenomenon. Here we describe a methodology that permits the study of two surface markers on lymphocytes conjugated to K562 target cells. In particular, the expression of low density CD8 (CD8 dim ) has been studied on both CD3 + and CD16 + lymphocytes bound to K562 target cells. Previously described methodologies, either optical microscopy or flow cytometry, were not able to identify the effector population by mAb double staining, especially in the case of antigens expressed at low density. The flow cytometric methodology described here permits the measurement of the binding activity of small lymphocyte subsets such as the CD3 + 8 dim+ population. However, the method could be used to study the binding activity of any effector population defined by mAb double staining.

Flow cytometry has been employed to establish an NK assay using the K562 target cell line. These cells show a perpendicular light scatter (PLS) characteristically different from lymphocytes. Other physical parameters, such as forward light scatter (FLS), do not discriminate between the two populations, since dead K562 cells display similar FLS characteristics as effector cells. Killed cells are stained with propidium iodide and followed by flow analysis. It was advisable to add the dye in the medium so that, as long as the target cells are killed they will also be stained. Moreover the flow cytometric and trypan blue evaluation of target cell death rate showed a stronger correlation than did either test with the conventional 51Cr release assay, the first two methods both being based on the same biological mechanism.

Morphological changes that occur in K562 cells after natural killing produce profound changes in cellular light scattering properties. The possibility of gating out all the effector cells by thresholding on perpendicular light scatter and the subsequent identification of two distinct clusters of cells, which correspond to dead and viable targets, have permitted the measurement of natural killer activity in vitro. The changes in scattering properties after cell death are mainly determined by the variation of internal refractive index of the dying cell. A comparison of the scattering and propidium iodide staining procedures showed good correlation. The morphological detection and measurement of cellular death is therefore used to estimate NK lytic activity. This methodology permits the measurement of NK activity without staining the target and the measurement of perpendicular light scatter provides an alternative approach to the study of lytic processes in vitro.

Peripheral blood mononuclear cells from ten healthy normal donors were analyzed by means of monoclonal antibodies and flow cytometry (FACS IV). In order to apply optimal gating for the identification and exclusion of monocytes from lymphocyte populations, mononuclear cells were analyzed on the scattergram using rectangular or poligonal computer-generated windows. Two main windows on lymphoid population were used which mainly differed for the up-right corner, in the border area between lymphocytes and monocytes. While the number of lymphocytes contained either in the regular or in the poligonal windows was the same, the number of contaminating monocytes decreased by two-fold in the poligonal one. Besides, the use of tight lymphocytes gating, in order to reach lower monocyte contamination, leads to a loss of lymphoid cells which does not appear to be random, but seems to affect mainly the Leu11c + population with natural killer activity. These cells produce forward and perpendicular scatter signals higher than other lymphocyte subsets, and, therefore, are mainly located in the area of the scattergram which divides lymphocytes from monocytes. These data are in accordance with the large granular morphology of natural killer cells. The use of the poligonal windows seems to be useful to reduce monocyte contamination with no selective loss of natural killer lymphocytes, and may be particularly helpful in the analysis of pathological samples.