Hi - it could be, that either you have contamination with cerebral membrane - or your pipettes are too big in diameter size. When I do raphe neuron culturing- I use blue pipette tips and poke holes with needles - large: 21G1 1/2 and small 26G 1 1/2. And you could try to cut cerebellum in smaller pieces before you put it in papain

Thanks Tina. I tried using P1000 following by P200 pipette tips and also fire polished pasteur pipettes. But neither method could solve the problem. May be I'll try cutting the tissue up before digestion.

Hi Pavithra- I think that P1000 and P200 are too big. With the fire polished pasteur pipettes I have no experience- and as far as I read- there was no size description in the paper. So what I did- I forced the liquid wit the tissue junks through those small holes. 

:-) Good luck 

Hi Pavithra

Try using 20 units/ml papain solution and incubate in papain solution at 37 degree centigrade for 30 min . I personally feel 45 min is too long a digestion time to get viable single cell suspension for neuronal culture. The paper you are referring to mentions use of trypsin to digest the tissue. Trypsin is a stronger enzyme at breaking down cellular adhesive proteins. There must be some reason for them using it to digest the cerebellum tissue. The extra cellular matrix could be different. Maybe higher activity papain solution and incubation will help. I do not know if you are adding cysteine when making the papain solution. It is needed for papain activity. Please do not forget to add DNase and I hope you are adding glucose too. Make sure the pH of the solution is around 7 (7.1 to 7.3) . And after the incubation please wash thoroughly with media containing serum (FBS). It helps halt digestion and keeps cells from being lost to lysis.

Do keep us updated. Best of luck

Shreya

Hi Shreya

Thanks so much for all your suggestions. I do add L-cysteine and EDTA to my papain activation buffer and incubate for 15 minutes before adding to the tissue and the buffer is adjusted to pH 7.1. Also, I use Ovomucoid to inhibit Papain after digestion. I am currently not using DNase, which may be causing part of the issue! I will try including this step and that may improve things. I will let you know how it goes next time.

Thanks!

Hi Pavithra,

I have cultured both mouse and rat neurons for many years, and adding DNase is very important when using enzymes to create single cell suspensions.  Your use of Ovomucoid is good--it is a better inhibitor of enzyme action than serum.  It is also important to remove the meninges and cut the tissue into smaller pieces before incubating in enzymes and dissociation by trituration.  You want to incubate the tissue for the least possible time, and then gently dissociate the material.

I noticed that you mention that you used polypropylene pipette tips for triturating your tissue. This can cause a lot of damage, which translates to sheared cells and the release of DNA.  While the polypropylene tips are more slick than glass, you are limited in the size of the opening.  In addition, not all tips have a nice edge, but can be a bit ragged.  Also, unless you are using a motorized pipetter at a very slow speed, it can be difficult to triturate the material slowly by hand, and not get bubbles in the mixture.

I suggest that you use Pasteur pipettes for trituration.  I use pipettes made of soda-lime glass rather than borosilicate glass so that it is easier to control the size of the opening.  I used to siliconize the pipettes, but now I just make sure to pull some of the trituration buffer with BSA into them first in order to keep the cells from sticking to the glass.

There are many helpful suggestions for neuronal cell culture in "Culturing Nerve Cells" edited by Gary Banker and Kimberly Goslin, and I have had good luck with several protocols in that book.  I don't have my copy available at the moment, but I believe there is a chapter on culturing cerebellar neurons.

Good Luck!

Jill

Hi again, Pavritha

The Greene lab has produced a JoVE video which shows how they isolate the cerebellar neurons using papain: Lee, H. Y., Greene, L. A., Mason, C. A., Manzini, M. C. Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons . J. Vis. Exp. (23), e990, doi:10.3791/990 (2009).  This is basically the same protocol as the paper from the Mason lab that you have linked here.

Since they are isolating granule cells here, they are using older animals than you may want for Purkinje neurons, but the enzyme preparation and trituration principal is the same.

The video says that they get the same dissociation using serum-coated P1000 tips as they get with glass pipettes.  However, if you are interested in trying glass pipettes, the 3 sizes of openings in the Trenkner protocol chapter in Banker and Goslin start at 1 mm and decrease to 0.25 mm.

I also suggest that you look at the original Baptista protocol in Neuron:

Baptista CA, Hatten ME, Blazeski R, Mason CA.  Neuron. 1994 Feb;12(2):243-60.  Cell-cell interactions influence survival and differentiation of purified Purkinje cells in vitro.

Cheers-

Jill

Is FBS sufficient for papain inactivation? Why/Why not?

Do I  really need to use Ovomucoid in my Papain Stop Solution?