By Peter J. Hollenbeck, James R. Bamburg
This e-book lays out a number of uncomplicated suggestions for becoming and conducting experiments with many sorts of neurons. matters contain peripheral and important neurons from vertebrate and invertebrate resources, in addition to neuron-like mobilephone strains. It additionally explains contemporary advances in our skill to introduce exogenous proteins and genes to neurons in tradition. techniques for winning protein infiltration, biolistic transfection, electroporation, and viral transgenic equipment in neurons also are offered. * comprises tradition method for greater than a dozen different types of CNS and PNS neurons * comprises most modern and trustworthy strategies from professional practitioners for particular experimental functions* Addresses the newest recommendations for transfecting neurons
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Extra info for Neurons: Methods and Applications for the Cell Biologist
2 ml of poly-d-lysine to the well. Two different methods can be used, one with a higher concentration and the other with a lower concentration of poly-d-lysine. The higher concentration generally provides better adhesion, but sometimes can produce toxicity, particularly if serum-free medium is used. If the latter proves to be the case, then the lower concentration should be used. Method 1: Coat the glass well with 1 mg/ml poly-d-lysine for 3 h at room temperature. Rinse the dish six times with sterile distilled water, 5 min each time.
In contrast, the two types of peripheral neurons are easier to culture, more rugged, survive injection, and can be grown under a much wider variety of culture conditions tailored to specific experimental needs. The reason why we use chick for sensory neuronal cultures and rat for sympathetic cultures is simply because of the backlog of knowledge from previous work. It is certainly possible to culture rat sensory neurons and chick sympathetic neurons, but fewer studies have been performed on these cultures, at least in our field.
Substrate One of the most important issues for neuronal cell culture is substrate. Neurons are poorly adhesive cells compared to many kinds of cells that can be cultured, and yet adhesion to the substrate is absolutely essential for their viability and their capacity to extend neurites. In general, neurons do not adhere to plain glass or even to plastic culture dishes (except when methylcellulose is added to the medium; see later). There are several possibilities for treating these surfaces that enhance their capacity to adhere to cells.