2.Protocol for preparing glial cultures(M.Aihara)
A.astroglia
needed materials:
dissociated brain cells
DMIT(-)/FCS10%
75cm2 flask
desirable plates (non-coating)
1. dissection and dissociation of cerebral cells (see the section of "protocol for preparing
hippocampal neuronal culture")
2. plate cells at the density of 1.0E7 / flask
3. replace culture media twice a week with fresh DMIT(-)FCS 10%
4. after 2 weeks, this culture should reach confluence with astroglia
note: if in a good condition, astroglia become confluent and on the surface or in medium,
microglia are present
5. shake the flask for a few minutes to detach microglia from the surface of the culture
6. ake sup to place in 50ml centrifuge tube (see next chapter "microglia")
7. wash this flask with PBS(-) 2 times and trypsinize cells with 0.25% trypsin for a few minutes
8. suspend them in DMIT(-)FCS10%
9. count cell density and plate in your desirable plates
note:you don't need to prepare coated plates because glia easily attach to plastic plates
10. after 24 hours from 2nd preparation, replace media with fresh one, for the purpose of removing neurons
or damaged cells
11. feed for 2 weeks replacing media twice a week
12. you can get astroglial confluent culture
B.microglia
continue from above chapter step 6
7. spin down cells for 5 min at 1000rpm
8. suspend pellets in DMIT(-)FCS 10%
9. count cell density and plate in your desirable plates
note:you don't need to prepare coated plates because glia easily attach to plastic plates
10. replace media after 6 hours with fresh one
11. assay should be performed within 2 days
note:isolated microglia become ameboid in a good condition but they cannot proliferate
note:astroglia and microglia are viable in serum free media (DMIT(-)BSA0.1%) for a few days