N2a cells differentiation - (Feb/15/2008 )
I need to differentiate N2a cells. Most of publications I went through recommends to do it in DMEM medium containing 5µM-5mM dibutyryl cAMP and 0-1% FBS for 48h (37C, 5% CO2).
I incubated N2a cells in DMEM with 2,5mM dibutyryl cAMP and 1%FBS for 48h, but after this time only very little cells exhibited a differentiated morphology with long neuritic processes (ca. 0,5%). Medium containing dcAMP and reduced FBS content was exchanged every 24h.
Could anybody recommend a good protcol for N2a cells differentiation? (medium composition, time, conditions of incubation in differentiating medium) or suggest possible reason why in case of my cells differentiation was so ineffective?
Description of dibutyryl cAMP says that it loses a dibutyryl group in solution at pH 8,5 which decreases its permeability through cell membranes. Is it possible that DMEM is getting alcalized in contact with air during changing the medium and then the dibutyryl group is lost?
Ceres
Incubation in serum free medium for 4-8 hours is sufficient for n2a to differentiate.
do you have any idea why my cells did not differentiated efficiently even after 48h?
how important is the density at which you plate the cells before you start differentiating them? What confluency would you say is recommended to let the cells form the processes?
Is that possible, that you can give me some more details about how are you dfferentiating N2a cels?
how important is the density at which you plate the cells before you start differentiating them? What confluency would you say is recommended to let the cells form the processes?
Is that possible, that you can give me some more details about how are you dfferentiating N2a cels?
I don't know. I transfected n2a with a gene which is supposed to facilitate its differentiation. However, serum removal which is included in the transfection protocol is sufficient to do the job.
I incubated N2a cells in DMEM with 2,5mM dibutyryl cAMP and 1%FBS for 48h, but after this time only very little cells exhibited a differentiated morphology with long neuritic processes (ca. 0,5%). Medium containing dcAMP and reduced FBS content was exchanged every 24h.
Could anybody recommend a good protcol for N2a cells differentiation? (medium composition, time, conditions of incubation in differentiating medium) or suggest possible reason why in case of my cells differentiation was so ineffective?
Description of dibutyryl cAMP says that it loses a dibutyryl group in solution at pH 8,5 which decreases its permeability through cell membranes. Is it possible that DMEM is getting alcalized in contact with air during changing the medium and then the dibutyryl group is lost?
Ceres
I've worked with a line very similar to N2a and we've usually used 1mM dbcAMP and 10% FBS supplemented media. However, it could also be done at 1% FBS without issues. The confluency would be kept relatively low to allow for neurite extension. We typically observed differentiation within at least 24 hrs. I would actually leave mine-- but what was to study phosphorylated neurofilaments.
Hope that helps.