I am trying to differentiate PC12 cells in 96 well plate ( 10000/ well) with the help of NGF but my cells kept on dividing even after the addition of NGFand forms a clump so they do not differentiate properly. Can anyone suggest a protocol with NGF?
Nitish,
You might have to serum starve the cells when you add NGF and do not change medium until it is differentiated completely. If you realize cells need more medium, just add more to the existing medium. Get down the serum to 2 % and add NGF containing medium while passage and adding cells into the 96 wells. Also do not shake the plate after adding cells for at least 20-30 min until all cell stick to bottom.
Good luck !
I suspect that concentration of NGF might be extremely low. I have recently estimated that the insulin concentration in the culture medium should be 0.36 mg/mL (file The Fascio effect), which might be c.a. 60-fold higher concentration than the concentration of a commercial supplier. Bovine insulin is surely a high cost hormone or drug (2800 USD/g), however making 10 mL of medium costs 10 USD and experiment may be possible to achieve.
However, NGF is surely high price at this time; i.e., 1000-fold higher price than insulin. Therefore, you would be better to purify independently NGF.
Beta-nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are rich in salivary glands (submaxillary gland、sublingual gland, and parotid gland) in order to maintain taste receptors in cranial nerves in the taste buds; i.e., saliva contains amylase, maltase and lipase, but does not contain protease and biotinidase to prevent injury of taste buds). Rat salivary glands may be good source. I would like to recommend the RP-HPLC method (please see file RP-HPLC lysozyme). NGF has Mr 13,494, alkaline pI of 9.0, and hydrophobicity 0.491, and BDNF has Mr 13,512, alkaline pI of 9.6, and hydrophobicity 0.462, which are very similar to lysozyme; i.e., Mr 14,313, alkaline pI of 11.35, and hydrophobicity 0.526. Elution order might be 1st BDNF, 2nd NGF, and 3rd lysozyme in accordance with hydrophilicity. Separation of these three proteins are possible to separate by improving the gradient programn or shape (see file Insuli RP-HPLC). This RP-HPLC method is the reliable protein determination method. Saliva and milk has many mucin-type large proteins. Therefore, acid extraction pretreatment for sample injection may be good.
Hi Nitish,
I have been trying to use NGF for differentiating PC12 cells and am having difficulties in differentating them. They are still dividing as you said. I have also tried using different NGF concentrations (50-200 ng/ml) and the cells still continue to divide. It seems that the cells begin to differentiate but I am not able to see long processes. Do you have any suggestions?
Thank you.
Khushboo.
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