Dissociation of CNS Tissue for Isolating Cells in EAE Mice

To begin, dissect the brain and spinal cord tissue and store the cell suspension on ice. Then prepare an appropriate volume of enzyme mix-1 and 2. Transfer 1, 950 microliters of enzyme mix-1 into the C tube, and add the tissue pieces of the CNS cell suspension.

Then add 30 microliters of enzyme mix-2 to the C-tube. Close the C-tubes tightly and attach them upside down onto the sleeve of the cell dissociator with heaters. Run the appropriate program and observe for at least five minutes to ensure all tubes turn at the same velocity.

In the meantime, place a 70-micron strainer on a 50-milliliter tube and pre-moisten the strainer with two milliliters of DPBS. After termination of the program, detach the C tubes from the dissociator and centrifuge the samples at 300 g for one minute at four degrees Celsius. Resuspend the sample and apply it to the pre-moistened strainer.

Add 10 milliliters of cold DPBS to the empty C-tube. Mix and add the suspension onto the corresponding strainer. After discarding the strainers, centrifuge the cell suspension again for 10 minutes, and then carefully aspirate the supernatant to obtain a cell pellet.