I am trying to isolate GFP +ve microglia from adult mouse brain (1-2mo). I have been using a modified protocol from Derecki (2012), but using a Percoll gradient for myelin removal.
My problem is that from a whole brain the total number of cells isolated for FACS is very low (approx. 1M) and that of these cells, many are dead/not cells but debris. The 50k-100k GFP +ve cells I get is not enough for a WB sample. Any help is much appreciated. I've summarised my protocol below:
- Remove brain and homogenise in HBSS 2-3 times with glass homogenizer
- Suspension put through 70um cell strainer into falcon
- Pellet filtered cells at 1200rpm, 4C, 10m with NO BRAKE
- Aspirate supernatant and resuspend in 30% Percoll in HBSS. Spin at 700g, 4C, 10m
- Aspirate supernatant and resuspend in 5mL HBSS at 1200rpm, 4C, 10m with NO BRAKE
- Resuspend pellet in 500uL FACS buffer and send for FACS
hi
As you said, the number of target cells is low, then you can increase the number of mouse . to achieve greater purity can also use the kit.
CD11b (Microglia) MicroBeads, human and mouse – small size
http://www.miltenyibiotec.com/en/products-and-services/macs-cell-separation/cell-separation-reagents/neural-cells/cd11b-microglia-microbeads-human-and-mouse.aspx
good luck
What is driving GFP expression in your model? In others words, are all microglia supposed to be GFP+? If not, then you won't get the entire population of microglia and using a kit may be your best option.
Alternatively, when we do our Percoll gradient we layer 30% Percoll over 70% and spin at 500g for 30 min. Then we take just the layer that forms between the two Percoll layers. You may not be getting enough separation of your layers to remove the debris and dead cells if you centrifuge for only 10 min and if you aren't careful about taking only the layer created by the gradient. You should see a layer of myelin/debris on the top of the gradient. You can also try a 40um cell strainer to help release more of the cells. Sometimes that helps, but not always.
Good luck!
Hi Sarah,
Thanks for your answer. The GFP is driven by the Iba1 promoter and therefore should label all microglia in the brain.
Your point about the 40um strainer is a really good one. When I spin in 30% Percoll I do get a very obvious thick white myelin band sitting on top. I think what may be happening is that the cells are not fully dissociated and getting pulled up with the myelin. I will try the 40um strainer and the 30/70 percoll layer and see if I get improved yields.
Many thanks,
James
Yes, if you are doing only 30% percoll and discarding the entire supernatant, keeping only the pellet as the source of your cells, you are actually getting rid of most of your microglia. That, more than the size of the strainer, would actually explain your cell loss.
Best of luck!
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