PC12 cell differentiation assay NGF is one of the earliest discovered neurotrophic factors. It is the most thoroughly studied neuron growth regulator with neuronal nutrition and pro-apoptotic growth. It is central to The development, differentiation, growth, regeneration and expression of functional properties of peripheral neurons have important regulatory effects.
1. Culture of PC12 cells: PC12 cells were cultured in a 7.5% CO2 saturated water vapor carbon dioxide incubator at 36 °C, and 2/3 of the culture medium was changed every 2 days. When the cells were close to confluence, the cells were cultured with the above culture medium containing 1 mmol/L EGTA. After 6 hours or more, the cells were washed 3 times with RPMI-1640/water (1:1), single cells were blistered, and culture was continued in 1:3 or 1:4 flasks. The doubling time of PC12 cells is about 2.5 to 4 days.
2. Detection of NGF activity:
1) PC12 cells were formulated into 106/ml using the above culture solution. 0.5 ml of cell suspension and serially diluted NGF samples or standards were added to each 35 mm dish. No NGF was added to the control dish. Incubate for 20 to 24 hours in a saturated water vapor carbon dioxide incubator at 37 ° C in 7.5% CO 2 .
2) PC12 cells were observed under a phase contrast microscope. The cells were clustered and counted into 100 cell clusters, in which the cell clusters with fibrillar cells were positive cell clusters. The dilution of the sample was plotted against the dilution of the sample, and the dose-response curve was plotted to calculate the amount of NGF in the sample.
NGF binds to the receptor, internalizes through receptor-mediated endocytosis, forms vesicles surrounded by the axon, contains NGF, and maintains its biological activity, and is transported retrogradely along the microtubules to the cell body through the axons. Transduction of a second messenger system such as tyrosine protein kinase, calcium acyl lactitol, endogenous cyclic adenosine, etc., initiates a series of cascades that regulate the expression of certain structural or functional protein genes of target cells. Play its biological effects.
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