I am trying to recover transgenic fluorescing expressing neurons from adult mouse brain. Papain dissociation system by Worthing was used in first few trials but yield no transgenic fluorescing neurons from FACS. I suspected that it could be due to harsh dissociation condition was applied and switch to digestion of brain section using Accutase at 4C for 30 minutes on a rotating mixer followed with trituration using P1000 micropipette and hold in Hibernate A medium supplied with BSA and Dnase I. However, I still recover non of neurons expressing the fluorescence signal. The population from FACS between WT and transgenic mouse brain shows no distinct difference in terms of the sorted population, indicating no fluorescing population.
So, advise on how to improve on brain dissociation of adult mouse brain is highly appreciated.
Dear Kit-yeng,
I don't use FACS, but I have tried to dissociate adult brain tissue. I have only used the Papain method, but I follow the Huettner-Baughman protocol in Culturing Nerve Cells, 2nd Ed, edited by Gary Banker and Kimberly Goslin. It uses specific buffers to keep the neurons healthy. It was also the basis of the Franke & Barres protocol for dissociating viable retinal ganglion cells. If your cells of interest are glutaminergic, it may have useful suggestions that can be adapted to Accutase dissociation.
One concern I have is that you say you are holding your cells in a media that includes the DNAse. I don't think that is a good idea. My experience is that you need to inhibit the DNAse as soon as you have the single cells.
I also urge care in the use of the P1000 for trituration. It is easy to kill cells if you triturate too quickly (bubbles are bad!) or through too small or rough of an opening. I had to purchase pipette tips with a wide opening, and I use a motorized pipettor set at the slowest speed. I also pre-wet the tip with the dissociation media before I triturate the tissue.
One suggestion for troubleshooting your protocol would be to look at an aliquot of your cells at each stage of your protocol using a fluorescent microscope so that you can figure out when the fluorescent neurons disappear. Are you able to microdissect out the brain region where they are present in large numbers? That might help speed up the dissociation.
Good Luck!
Jill
Jill Miotke
Thank you for your reply, highly appreciate it!
I think I am using the same Papain method that you mentioned previously but gotten bad result. According to Worthington, the protocol was adapted from the same source that you have quoted. (http://www.worthington-biochem.com/pds/default.html)
Perhaps, it could be the problem with the gas equilibration with 95% O2 and 5% CO2. How did you perform this step to maintain the physiological pH of the buffer for holding the brain tissue throughout the whole dissociation process?
Oh, I thought the presence of BSA would be good to help maintain the viability of the neuron cells when DNase is present. I will stop using this holding media and just simply hold in Hibernate-A medium.
Thanks.
Kit-yeng Sheng have you figured out how to do this? I am looking to do adult neuronal cell culture and would like to see how you progressed along with if you have any tips for me?
Thanks!
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