Removal of Debris and Red Blood Cells from CNS Cell Suspension of EAE Mice

To begin, obtain a cell pellet after dissociation of CNS tissue and re-suspend it with 3, 100 microliters of DPBS in one 15-milliliter tube. Do not vortex. Add 1, 800 microliters of the debris removal solution from the adult brain dissociation kit to two pooled CNS cell suspensions.

Invert the tube and mix the suspension, then gently overlay it with 4 milliliters of cold DPBS and observe a clear gradient. Centrifuge the tubes for 10 minutes at 3, 000g at 4 degrees Celsius with full acceleration and no break. After centrifugation, three phases are formed.

Aspirate the two top phases completely and discard them, ensuring no myelin residues are left behind. Fill up the tube with cold DPBS up to 14 milliliters and close it. Invert the tube with force on the workbench until the cell pellet becomes detached from the bottom of the tube.

Centrifuge the sample and aspirate the supernatant completely. For red blood cell removal, add 1 milliliter of the red blood cell removal solution to the cell pellet. After re-suspending the pellet, incubate the solution for 10 minutes at 4 degrees Celsius.

Then add 10 milliliters of cold PB buffer, centrifuge the sample, and completely aspirate the supernatant. For cell counting, dilute the cell suspension by 50 times in PB buffer and then by 1 to 10 dilution in 0.4%Trypan Blue solution. Then determine the cell count, using an improved counting chamber.