Thursday, January 12, 2012

Isolation of rats microglial cells

Microglial cells are found throughout the central and peripheral nervous system. Microglial cells express low levels of MHC class I antigen and they respond to injury.

The following protocol describes the isolation of rat microglial cells. However due to the low yield several mice or rats should be sacrificed.

Method:

1) Sacrifice the rats by cervical dislocation or ether overdose.
2) Perfuse each animal via the carotid artery with 250 ml cold sterile PBS solution. The central nervous system must be completely perfused.
3) Remove the brain and spinal cord and transfer the organs to a sterile petri dish containing cold sterile HBSS (Invitrogen) supplemented with 3% fetal calf serum.
4) Mince the brain and spinal cord. (Work under a sterile hood)
5) Pass the tissue through a sterile stainless steel mesh into a sterile petri dish containing cold HBSS (Invitrogen) supplemented with 3% fetal calf serum.
6) Pour the tissue and buffer into centrifuge tubes and centrifuge the tubes in refrigerated table-top centrifuge at 4 degree centigrade for 15 minutes.
7) Digest each brain/spinal cord for 90 minutes at 37 C in 1.5 ml of dissociation buffer (Dissociation buffer: 42 mM MgCl2, 23 mM CaCl2, 50 mM KCl, 153 mM NaCl, 0.75% collagenase type II (Sigma), 7000 U/ml DNase I (Sigma)
8) Add 5 ml of HBSS with 3% fetal calf serum.
9) Centrifuge the tissue and buffer mixture at 300 g to the pellet the cells.
10) Resuspend the cells in Percoll at 1.098 g/ml diluted in HBSS (final density 1.088 g/ml). See the protocol here: http://clodrosome.blogspot.com/2012/01/percoll-continuous-density-gradient-for.html and separate the microglia from other cells by centrifugation.

For more information about macrophage depletion visit www.clodrosome.com

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