The assembly of synapses in the vertebrate central nervous system requires bidirectional signaling across the synaptic cleft that directs the differentiation of pre- and postsynaptic membrane domains. Biochemical and genetic studies have identified several adhesion and signaling molecules that localize to synapses and might participate in organizing synaptic structures. Understanding how individual proteins contribute to synaptic organization is complicated by the fact that there are significant numbers of separate signals that cooperate in this process. This protocol describes an assay system that permits examination of synaptogenic activities of individual cell-surface proteins in isolation. Besides the time needed for preparation and growth of primary neuronal cultures (6-14 days), the execution and analysis of the assay is rapid, requiring approximately 2 days. Using this assay, recent studies revealed that single synaptic adhesion complexes can direct a remarkable degree of synaptic differentiation and provided new insights into the cell biological mechanisms of synaptogenesis.
Saturday, 2 June 2007
How to Prepare a copper-based fluorescent probe for nitric oxide and use it in mammalian cultured cells
A procedure for the preparation of a copper(II) complex (CuFL) as a fluorescent nitric oxide (NO) detector is described. The fluorescein-based ligand FL can be synthesized in seven reaction steps (overall yield ?20%), typically requiring a total time of 9 days. The CuFL probe allows for the detection of NO produced in mammalian cultured cells. The detailed protocol for the use of CuFL for imaging NO in human neuroblastoma SK-N-SH cells takes a total time of ?26 h. This includes plating cells on six-well tissue culture plates or imaging dishes, treatment with CuFL, stimulation of NO synthases and imaging by fluorescence microscopy.
Friday, 1 June 2007
Generation of functional hemangioblasts from human embryonic stem cells
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http://www.nature.com/nmeth/journal/v4/n6/pdf/nmeth1041.pdf
Thursday, 31 May 2007
Freezing and Thawing Eukaryotic Cells
From Dr. Bart Frank Library, Arthritis and Immunology Program, OMRF
Freezing cells in liquid nitrogen
For T25 Flask
- Take off supernatant
- Trypsinization with 1ml 0.25% EDTA
- Give a hard Shock to the flask to remove all attached cells
- Add 10ml Media +10%FCS
- Pipette up and down to distribute cells throughout media (i.e. not clumped together)
- Add resuspended cells to sterile universal tube
- Spin down 1500rpm, 3 mins
- Take off media
- Resuspend pellet in 2ml FCS +10%DMSO
- Distribute in 500ul aliquots (0.5 to 2 Million cells/ml)
- Move cells to -80oC overnight in Mr. frosty box (filled with ethenol)
- Finally freeze cells in liquid N2
Unthawing
- Warm DMEM in waterbath
- Thaw cryovial at 37oC quickly until cells become molten.
Aliquot the 1ml of cells using a disposable pipette into 10ml fresh media in a TC flask