Dear all,
i have been trying to isolate microglia of adult murine brain . i used a percoll density gradient; unfortunately it worked only in the first time for me, very well. after that.. it failed. no layers visible. no cells at the aimed position in the gradient.
#i would be very happy if you could share your protocols with me. i need a simple method in order to get macrophages of a brain.
#hope you can help.
regards
silke
Hi Silke,
I have recently been troubleshooting adult microglia isolation from rats using Percoll and had a similar experience as yours (success on the first and then much lower yield on the next attempt). What I found fixed the problem was using much gentler/slower mechanical homogenization of the brain, and then filtering through a 100um cell strainer first prior to 70um.
Hope this is of some help to you!
Austyn
this is awesome, thank you !! i will try this out asap. hopefully it works, thanks again!!
Dear Austyn,
can you tell me how many mice you have used, and then put in put in one tube and in which volume..... maybe even share your protocol with modifications?
i still struggle...
best
silke
thank you all for your support and the protoco,, Austyn!!actually,
i had tried twice again and i guess found the mistake/solution...
apparently one has to make very carefully the layering, i watched some youtube videos.. so when pouring the different percoll percentages, one has to tilt the tube nearly horizontally, so that the two different gradients meet each other like two currents from two directions, very slowly....
it is all about that step.
after this... it has worked so nicely...
hope this also helps you:)
Dear Silke,
in case you still have some troubles with microglia isolation using different percale percentages, you could also try to isolate the microglia using Miltenyi Kits (Neural tissue dissociation Kit P) followed by 30% percoll and Macsing. More details you can find in our publication here:
https://www.researchgate.net/publication/341788523_The_TSPO-NOX1_axis_controls_phagocyte-triggered_pathological_angiogenesis_in_the_eye
This worked for us pretty well and is avoiding the tricky percoll layering.
All the best,
Anne