This protocol describes how to prepare a single-cell suspension from harvested mouse brain tissue prior to downstream isolation of microglia using the EasySep™ Mouse CD11b Positive Selection Kit II.

Materials

• EasySep™ Mouse CD11b Positive Selection Kit II (Catalog #18970)
• Papain (Catalog #07465)
• DNase 1 Solution (1 mg/mL, Catalog #07900)
• HBSS, Modified (Without Ca++ and Mg++; Catalog #37250) containing 2% FBS and 1 mM EDTA OR DMEM/F-12 with 15 mM HEPES (Catalog #36254) containing 2% FBS
• 30% Percoll solution
• Scalpel
• 37 µm reversible strainer, large (e.g. Catalog #27250)
• 100 mm tissue culture-treated dish (e.g. Catalog #38046)
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• 14 mL round-bottom tube (e.g. Falcon® Round-Bottom Tubes, Catalog #38008)
• 50 mL conical tube (e.g. Falcon® Conical Tubes, Catalog #38010)
• Pipettor (e.g. Corning® Lambda™ Plus Pipettor, Catalog #38060)
• Pipette tips (e.g. Corning® Filtered Pipette Tips, Catalog #38034)
• Serological pipette controller
• Serological pipettes (e.g. Falcon® Serological Pipettes, 10 mL, Catalog #38004)
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Protocol

Part I: Mechanical Digestion of a Mouse Brain Sample

1. Prepare 3 mL of brain dissociation medium for up to 3 brains. For 4 or more brains, prepare 1 mL of brain digestion medium per brain. Add Papain (Catalog #07465) to a final concentration of 20 units/mL, DNase I Solution (1 mg/mL, Catalog #07900) to a final concentration of 100 μL/mL, HBSS, Modified (Without Ca++ and Mg++; Catalog #37250) or DMEM/F-12 with 15 mM HEPES to make up the remaining volume. Warm medium to room temperature (15 - 25°C) before use.
   Note: Activate Papain immediately before use. To ensure full activity, incubate reconstituted Papain in a solution containing 1.1 mM EDTA, 0.067 mM mercaptoethanol, and 5.5 mM cysteine-HCl for 30 minutes.
2. Prepare 100 mL of sample preparation medium: DMEM/F-12 with 15 mM HEPES (Catalog #36254) containing 2% FBS, or HBSS, Modified (Without Ca++ and Mg++; Catalog #37250) containing 2% FBS and 1 mM EDTA.
3. Perform dissections on the CNS tissue region of interest from adult mouse or rat brains and transfer dissected tissue pieces to the 100 mm dish. Otherwise, transfer freshly-harvested brains to a 100 mm dish without medium.
4. Transfer freshly harvested brains to a 100 mm petri dish containing 1 mL of brain dissociation medium.
5. Mince into a homogenous paste (< 1 mm in size) using a razor blade or scalpel.
6. Transfer the minced brain tissue to a sterile 50 mL conical tube. Rinse the dish with the remaining dissociation medium, and then add it to the 50 mL conical tube.
   Note: Avoid introducing air bubbles into the tissue suspension during transfer steps.
7. Incubate the minced tissue at 37°C for 30 minutes on a shaking platform.

Part II: Preparation of a Single-Cell Suspension

1. Place a 70 μm nylon mesh strainer over a new 50 mL conical tube. Rinse with sample preparation medium.
2. Transfer the disassociated brain tissue into the strainer. Push the digested brain tissue through the strainer with the rubber end of a syringe plunger to obtain a cell suspension. Rinse the strainer with more sample preparation medium.
   Note: If the strainer becomes clogged with brain tissue, transfer the contents to a new, pre-wetted filter.
3. Centrifuge the suspension at 300 x g for 10 minutes with the brake set to low.
4. Carefully remove and discard the supernatant using a serological pipette or aspirator.
5. Gently tap the tube to disassociate the cell pellet.
6. Add 6 mL per brain of 30% Percoll solution to the pellet. Mix gently and transfer to an appropriate-sized tube.
   Note: For volumes greater than 30 mL, use 50 mL conical tubes. For volumes less than 30 mL, use 1 or more 14 mL tubes.
7. Centrifuge at 700 x g with the brake off.
8. Carefully remove and discard the upper myelin layer using a 1 mL wide-bore pipette tip or a 2 mL serological pipette.
9. Remove and discard the remaining supernatant using a serological pipette.
10. Transfer the cells to a new tube, and top up with the sample preparation medium.
11. Centrifuge at 300 x g for 10 minutes with the break on low.
12. Carefully remove and discard the supernatant.
13. Gently tap the tube to disassociate the cell pellet. Cells are now ready for downstream use.