Isolation of Multiple Cell Types from a Mouse Brain by Mechanical Homogenization

Protocol

Disclaimer:  All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Mechanical homogenization of the brain and spinal cord

NOTE: Volumes described in this section are enough for the homogenization of one-half brain or spinal cord.

1. Pre-chill the glass mortar of the Dounce tissue grinder (Table of Materials) set on ice.
2. Add 3 mL of pre-chilled 1x Hank's balanced salt solution (HBSS) to the mortar.
3. Transfer the tissue (brain or spinal cord) from the well of the 6-well plate into the glass mortar, making sure it is dipped in 1x HBSS and sits at the bottom of the mortar.
4. Gently smash the tissue with 10 strokes of pestle A followed by 10 strokes of pestle B. Transfer the homogenized mix into a new 15 mL conical tube.
5. Fill the tube to a final volume of 10 mL by using pre-chilled 1x HBSS and centrifuge for 10 min at 320 x g at 4 °C.
6. Aspirate the supernatant and add ice-cold 1x HBSS to each tube up to a final volume of 7 mL and gently resuspend the pellet by vortexing.

2. Debris removal

NOTE: Removal of debris, composed mainly of undigested tissue and myelin sheaths, is a critical step to allow efficient staining of the tissue homogenate for subsequent flow cytometric analyses.

1. Filter each sample through a 70 µm cell strainer to remove any undigested tissue chunks. This step is particularly important when working with spinal cord tissues, as these samples are more likely to contain undigested nerve fragments or meninges that could affect the subsequent steps.
2. Make sure that the final volume is 7 mL in each sample tube. If not, fill with ice-cold 1x HBSS up to 7 mL.
3. Add 3 mL of pre-chilled isotonic Percoll solution (IPS) to each sample to make a final volume of 10 mL of a solution containing density gradient medium at a 30% final concentration. Gently vortex the samples to ensure they are homogeneously mixed.
4. Centrifuge samples for 15 min at 700 x g at 18 °C, making sure to set the acceleration of the centrifuge to 7 and the brake to 0.
   NOTE: Centrifugation should take approximately 30 min.
5. Delicately remove the samples from the centrifuge.
   NOTE: A whitish disk composed of debris and myelin should be visible floating on the surface of the solution. A pellet (containing the cells of interest) should be visible at the bottom of the tube.
6. Carefully aspirate all the whitish disk of debris and then the rest of the supernatant, making sure not to dislodge the pellet. Leave about 100 µL of solution on top of the cell pellet to avoid the risk of inadvertently dislodging it.
7. Add 1 mL of flow cytometry (FACS) blocking (BL) solution, resuspend the pellet by pipetting up and down with a 1000 µL pipette tip, and transfer samples to 1.5 mL tubes.
8. Centrifuge for 5 min at 450 x g at room temperature (RT).
9. Gently aspirate the supernatant and resuspend the pellet in the appropriate buffer compatible with downstream analyses

Materials

Name	Company	Catalog Number	Comments
10X HBSS (Calcium, Magnesium chloride, and Magnesium Sulfate-free)	Gibco	14185-052
70 mm Cell Strainer	Corning	431751
Conical Tubes (15 mL)	CellTreat	229411
Conical Tubes (50 mL)	CellTreat	229422
Dounce Tissue Grinder set (Includes Mortar as well as Pestles A and B)	Sigma-Aldrich	D9063-1SET
Percoll	GE Healthcare	10266569	sold as not sterile reagent
Percoll	Sigma	65455529	sterile reagent (to be used for applications requiring sterility)
Percoll PLUS	Sigma	GE17-5445-01	reagent containing very low traces of endotoxin