Technical Reference #1854

1854.

Cdk5 Nuclear Localization Is p27-dependent in Nerve Cells IMPLICATIONS FOR CELL CYCLE SUPPRESSION AND CASPASE-3 ACTIVATION* Jie Zhang; Huifang Li and Karl Herrup, Rutgers State University of New Jersey, The Journal of Biological Chemistry, 285(1854), (2010)
Link To Paper

Keywords:
Cell/Neuron; Cell/Cycle; Cell/Cyclins; Cell/Division; Diseases/Alzheimer Disease Neurodegeneration; Nuclear Export; Nuclear Import; Nucleocytoplasmic; p27

Materials & Methods:
For Cdk5-deficient cultures all of the embryos from a Cdk5 / Cdk5 / mating were harvestedand treated separately. Isolated embryonic day 16.5 embryonic cerebral cortices from control Cdk5 / or E2f1 / were treated with 0.25% trypsin-EDTA and dissociated into single cells by gentle trituration. The cells were suspended in neurobasal medium supplemented with B27 and 2 mM glutamine and then plated on coverslips coated with poly-L-lysine (0.05 mg/ml) and laminin (5 g/ml). All of the cultures were grown for a minimum of 5 days in vitro before treatment. To monitor cultures during treatment Cdk5 / or wild type neurons were cultured in glass-bottomed culture chambers (Mat-Tek Corp.). After transfection or drug treatment the dish to be monitored was placed into a CO2 and temperature-controlled chamber mounted on the motorized stage of an inverted microscope (Leica LTM). Multiple neurons were monitored simultaneously using IP Lab software (BD Biosciences CA). GFP and DsRed were visualized with L5 and N3 filter sets respectively.

Microscopic Technique
Inverted Microscopy

Cell Type(s)
Neuroblastoma (Neuro- 2a; N2a) and NIH 3T3 cells