In vitro assay for the quantitation of apoptosis-induced alterations of nuclear envelope permeability.
This protocol describes how to perform comparative measurements of the permeability of the nuclear envelope in adherent cells. The plasma membrane is permeabilized at low digitonin concentrations, leaving the nuclear membrane intact. These semi-permeabilized cells are incubated with cytosolic extracts prepared in advance and with a fluorescent reporter molecule whose molecular weight exceeds the size-exclusion limit of the nuclear envelope. Images are taken with a confocal microscope and subsequently analyzed using a custom-made software program that recognizes the nuclei automatically and calculates the mean nuclear fluorescence signal. Here, we measure the increase in nuclear permeability triggered by cytosolic extracts from cells dying by apoptosis. This method can be employed for the study of processes that affect the nucleocytoplasmic distribution of fluorescent molecules in cell populations. The large size of the samples means that subtle fluctuations in nuclear fluorescence can be detected with a high confidence level. Isolation of cell extracts takes 5-6 h, and the preparation and imaging of 15 or so specimens takes 4-5 h.
Grote
Patricia
P
Department of Biology, University of Konstanz, Universitaetsstrasse 10, D-78464 Konstanz, Germany.
Ferrando-May
Elisa
E
D017209:Apoptosis / Q000502:physiology*; D006801:Humans; D019169:Jurkat Cells; D018613:Microscopy, Confocal; D015336:Molecular Probe Techniques; D009685:Nuclear Envelope / Q000378:metabolism*; D010539:Permeability