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 up-regulation 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.
A flow cytometry procedure for simultaneous characterization of cell DNA content and expression of intracellular protein kinase C-zeta.
BERTAGNOLO, Valeria
2006
Abstract
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 up-regulation 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.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
