I have a problem with transfecting isolated primary mouse microglia. I isolated the primary MG by shaking, and the problem is that there is low concentration of MG.
It is difficult to transfect it with a low concentration. Any suggestions/advice is much appreciated.
Because these cells do not proliferate, lenti and adenovirus are the best options
Hi, I have the same problem as you. We have very low concentration of pimary mouse microglia cells. We seed 50.000 cells in a 24 well plate. Then we use lentiviral particels to transduce microglia cells.
Hi Sandra, thank you for your comment; Have you tried it also with humany microglia, and how did you isolate the microglia (in which method)?
Thank you for your response
Ed
Hi Eduard,
no we have no human microglia. We isolate cerebral cortices and mesencephali from 2-3 old mice/rat pups. Microglia were collected as free-floating cells from primary cultures of astrocytes and cultivated as described in Wilms et al. I attach one of our paper.
Sandra
Hi guys, I used to use the mild trpysination method to isolate primary microglia. Sometiomes it worked well,but mos time I have the same problem as you_the yield was very low. And after I obtain the nearly purified microglia, I would not reseeding again to avoid further stimulation. So the cells did not repond quite the same. Maybe the number is a major problem.
I am wondering how do you solve these problems regarding the yield and the reseeding? Thanks very much~~~~
Hey Yuan, how many animals do you use for experimants or for each well for isolating microglia?
Ed
I have found this protocol to yield far more microglia than shaking mixed cultures. Please see http://www.jove.com/video/50079/isolation-and-culture-of-mouse-cortical-astrocytes. Essentially, you culture the mixed glia until confluent, then add a dilute trypsin mix and the astrocytes just come off in a sheet, leaving unperturbed microglia. It works great.
I just saw that Yuan Li does this as well. I feel like I get more microglia the longer I let the mixed glial cultures go for. Once the microglia are isolated, they don't proliferate much further. You can also combine this with the lidocaine protocol that removed microglia from the top of the mixed glial culture - http://www.jove.com/video/50647/culturing-microglia-from-the-neonatal-and-adult-central-nervous-system
Has anyone had success with Lipofectamine? It is killing my cells. I can't tell if it is working at low concentrations since my starting number of cells is so low to begin with, nothing is getting transfected.
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