||100 mL basal medium plus Supplement A, Supplement B and Supplement C
||Blue ice - basal medium, Dry ice - Supplements A, B and C
||100 mL basal medium should be stored at 4oC, Supplement A, B and C should be stored at -80oC
||Supplement the medium before use with Supplement A, Supplement B and Supplement C
Frequently Asked Questions
We are in Massachusetts. How long does it take for shipping the cells?
It would likely take 2 days for the cells to reach you. We have tested a 3-day shipment of plated microglia and microglial precursors from the UK to the US with success, so this should be fine.
Can I do ICC and flow cytometry with Axol microglial cells?
Given you would like to do ICC and flow rather than assays in 96 well plates, it would make sense to receive precursor cells that you can plate out onto your format of choice and then differentiate yourself. For flow cytometry, we could seed T25 or T75 flasks for you and send you the flasks, which will give you cells to fix and stain.
Do you test the microglia after 21 days? How long could it be kept?
Assays are recommended between 14-21 days as this is when the cells:
a) are fully differentiated (ie. maximally express microglia signature genes)
b) fully mature (e.g. have most ramified morphology, respond best to stimulation etc.).
After the 21 day period, the cells become unhealthy and start to die off, which will affect cell number and stop an assay working. In co-culture, the cells are able the survive longer due to support from the neuronal layer, but optimally experiments should be run during the 7 day window
Any suggestion for detachment reagent?
The cells cannot be detached for passaging. They can be detached for flow cytometry, as long as the cells are fixed immediately before they have a chance to become activated (within 30 minutes). One of our collaborators used Detachin to lift the cells for FACS analysis, as this is a quick and gentle treatment.
We would like to detach and re-seed the microglia. What kinds of pretreated cover glass (or plate, flask) do you suggest?
1) The microglia once plated cannot be passaged because:
a) they are difficult to detach without scraping as they adhere strongly and
b) they will consequently become activated as a result of this passaging once replated into the new vessel.
It is for this reason we have been working towards ways of shipping the undifferentiated microglial precursors to customers to give them more flexibility with regards to plate format, seeding into co-cultures etc.
2) Our microglia are seeded onto TC-treated plastic, no coating reagents are required. The adherence to the plastic actually drives the differentiation. As for glass, we believe they are able to adhere to it, however we have not tried this in-house at Axol with our cells, so cannot comment. Other groups have reported success coating with growth factor reduced Matrigel, but poly-D-lysine, for example, might also work.
Do these microglial cells proliferate after I receive it?
No, the microglial precursor cells seeded at Day 0 are non-proliferative and the cell number remains stable over the 2-3 week differentiation.