I found in RG  an answer of Marc Watson. Please have a look his answer below.

"In spite of your original attempts, I would overwhelmingly recommend papain for brain tissue digestion (particularly embryonic/postnatal). Over the last 13 years I've done mechanical, trypsin, papain, you name it! Papain easily outperforms everything for both neuronal and glial cell preparation. With neurons you can get near 95-100% viability and mixed glial preps establish much better, and faster. Papain is particularly good for getting good yields of OPCs I've found. i recommend you get the Papain Dissociation Kit from Worthington and use this as instructed. It works extremely well! Another little tip - chopthe brain tissue with a sharp, sterile scalal before digestion to increase the surface area and allow easy and gentle dissociation. Hope this helps Rebecca, please just ask if you've any more questions!"

Please, open in the attachment some papers and protocols. All the best.

Vladimir

I found in RG  an answer of Marc Watson. Please have a look his answer below.

"In spite of your original attempts, I would overwhelmingly recommend papain for brain tissue digestion (particularly embryonic/postnatal). Over the last 13 years I've done mechanical, trypsin, papain, you name it! Papain easily outperforms everything for both neuronal and glial cell preparation. With neurons you can get near 95-100% viability and mixed glial preps establish much better, and faster. Papain is particularly good for getting good yields of OPCs I've found. i recommend you get the Papain Dissociation Kit from Worthington and use this as instructed. It works extremely well! Another little tip - chopthe brain tissue with a sharp, sterile scalal before digestion to increase the surface area and allow easy and gentle dissociation. Hope this helps Rebecca, please just ask if you've any more questions!"

Please, open in the attachment some papers and protocols. All the best.

Vladimir

Hello Vladimir,

Thank you so much for your response. I currently use papain from Worthington to dissociate my tissue but after FACs sorting I found that my single cell preps had a large amount of debris like myelin. I know there are magnetic columns which remove myelin and I know of a method which uses a sucrose gradient to parse out cells from debris, but I was wondering if there were any other methods out there.

Best,

Michelle

I agree with Kulchitsky and the Watson ref, Papain will help address this issue.

Also, no matter the dissociation approach, it is often helpful to simply rinse with PBS and refresh your growth medium (hours after plating when cells have adhered, but within the same day-zero). This will aid the removal of unwanted debris.  If you are isolating a glial cell type, then you can first forcefully agitate the debris away from the cells/plate by lightly smacking your plate against the bench a few times before rinsing (don't agitate too hard, as you might lose some OPCs and MG).  Glial isolations tend not grow as well unless this simple step is performed.

Good Luck,

Ben

Thank you Benjamin.

Thank you Michelle for answer.

Thank you Benjamin and Vladimir!