Sequential CNS population isolations

Equipment/Supplies/Reagents

• 24-well plates (this protocol is intended for 24 well plate size, important for cell density considerations)
• 12mm microscope cover glass (Radiacwash treated to remove contaminants, stored in 100% EtOH)
• Bunsen burner (flame polish cover glass)
• Centrifuge (capable of 300 rcf, cooling to 4°C, and holding 15/50 mL tubes)
• Water bath shaker (optional, swirling every 5 minutes just as effective)
• Carbogen bubblers (Meduna’s Mixture, 95% O2: 5% CO2)
• Brain dissection equipment (ice stage, petri dish, forceps, surgical scissors)
• Pipettes (various sizes), pipette aid, serological pipettes, fire-polished Pasteur pipettes
• 0.2 um sterile syringe filter and syringe
• 70 um sterile cell strainer
• Hemocytometer
• Mouse pups (P2-3 optimal, up to P7 can be used)
• Poly-ornithine (0.1 mg/mL; sigma-aldrich, P3655-100mg)
• Laminin (sigma-aldrich, L2020-1MG )
• Worthington Papain Dissociation Kit (Worthington Biochemical, LK003153)
• ACSF or neuron dissection media (+hepes)
• 0.5% bovine serum albumin in phostphate buffered solution (0.5% BSA/PBS; 290-300 osmol)
• Anti-CD11b+ microbeads (Miltenyi biotec, 130-093-634)
• Anti-myelin microbeads (Miltenyi biotec, 130-096-733)
• ACSA-2 Microbead kit (Miltenyi biotec, 130-097-678)
• Serum-free astrocyte media (see recipe)
   

Prep work:

• Sterilize coverslips with Bunsen burner and add to plates
  • (if no Bunsen burner: add to plate, let EtOH dry 10-15 mins)
• Incubate with 500uL poly-ornithine (0.1 mg/mL; sigma-aldrich, P3655-100mg) for 2-24hr room temperature
  • Poly-o found in culture room freezer
• Wash coverslips with sterile H20 x3
• Let dry open until no droplets are visible
• Add 1uL laminin (sigma-aldrich, L2020-1MG ), swirl with flattened pipet tip to cover entire coverglass
  • Laminin found in culture room freezer in orange box
• Place serum-free astrocyte media (see recipe) in water bath to warm.
• Place Worthington Papain Dissociation Kit (Worthington Biochemical, LK003153) items on ice for preparation

Worthington Papain Dissociation:

1. One Time Only: Add 32mL of EBBS (vial 1) to the albumin-ovomucoid inhibitor mixture (vial 4) and allow the contents to dissolve. 
2. Add 5mL EBSS (vial 1) to papain vial (vial 2). Mix gently to dissolve papain.
3. Add 500μL of EBBS (vial 1) to a DNase vial (vial 3). Mix gently and transfer 250μL of this solution to the vial containing the papain. Store the rest of the DNase solution on ice/in fridge. 
   1. I add DNase using 200 pipet at 125ulx2 (1000 tips don’t fit well in tiny bottle)
4. Bubble papain with 95O2:5CO2 during dissection, keep at RT
5. Dissect cortex in bubbled ACSF (or brain region of interest)
6. Remove papain solution from bubbling, and transfer the mixed papain/DNase solution into a 50mL conical tube
   1. Sterile filter papain using syringe and 0.2 um filter before adding tissue
7. Take the cortical segments and place them in a petri dish with bubbled ACSF. Mince the tissue into small pieces. 
8. Draw the minced tissue using a pipette (on S) and let the tissue separate from the solution. Release only the settled tissue into the papain/DNase solution. 
9. Turn the shaker on in the hot water bath and incubate the tube containing the tissue for:
   1. 10-15 mins
   2. Swirl tissue in tube every ~5mins to allow maximum papain exposure and dissociation
10. After the incubation period, titrate the mixture with 10mL pipette gently with pipette setting turned to Small for 3-5x up and down 
11. Centrifuge the homogenized solution at 300*rcf for 5 min at room temperature. 
12. During this time, prep the resuspension media and density gradient:
    1. mix 2.7mL EBSS (vial 1) with 300μL reconstituted albumin-ovomucoid inhibitor (vial 4) and 150μL DNase solution (vial 3) into a 15 mL conical tube
    2. add 5 mL of the albumin (vial 4) to a 50 mL conical tube
13. Discard the supernatant and immediately resuspend the cell pellet in 1mL resuspension media
    1. Titruate on S, 5-7x up and down using fire-polished glass pipet to create single-cell suspension
    2. Add remaining volume of resuspension media 
14. Carefully layer the cell suspension on top of albumin, then centrifuge at 300*rcf for 3 min. at room temperature. 
15. Discard the supernatant and immediately suspend the pelleted cells in 5 mL 0.5% BSA in PBS (referred to as buffer). Filter the solution using 70μm BD Falcon filter to remove any non-dissociated tissue.
    1. Wet the filter before use
    2. I usually use 1mL to wet the filter, then add 4mL to the pellet, then apply to filter

ACSA-2 Antibody-Microbead pull down

1. Centrifuge solution at 300*rcf for 3mins at 4C; discard supernatant
2. Resuspend pellet in 150uL buffer
3. Add 10-15 uL Anti-CD11b+ microbeads (Miltenyi biotec, 130-093-634)
4. Add 10-15 uL Anti-myelin microbeads (Miltenyi biotec, 130-096-733)
5. Incubate 10 mins at 4C, mixing every ~5 minutes
6. “Wash” by adding 1mL 0.5% BSA/PBS (“buffer”)
7. Centrifuge at 300*rcf for 3 mins at 4C, discard supernatant
   1. This will remove any excess beads from solution
   2. While centrifuging, prep for magnetic separation
      1. Put LS column in holder, wash 2mL buffer through x1
8. Resuspend pellet with 500uL buffer, and apply directly through column
   1. Collect the flow through (this is the fraction of cells that were not labeled, contains astrocytes + neurons)
9. Wash the column with 3mL buffer, x2
   1. Continue to collect the flow through
   2. You can discard the column after washes (should only have microglia + myelin)
10. Centrifuge flow through at 300*rcf for 3 mins at 4C, discard supernatant
11. Resuspend pellet in 150uL buffer
12. Add 10-15uL FcR blocking buffer from ACSA-2 Microbead kit (Miltenyi biotec, 130-097-678)
13. Incubate 10 minutes at 4C, mixing every ~5 minutes
14. Add 10-15uL ACSA-2 microbeads from kit (Miltenyi biotec, 130-097-678)
15. Incubate 10 minutes at 4C, mixing every ~5 minutes
16. Add 1mL buffer to “wash”
17. Centrifuge at 300*rcf for 5 mins at 4C, discard supernatant
    1. This will remove any excess beads from solution
    2. While centrifuging, prep for magnetic separation
       1. Put LS column in holder, wash 2mL BSA/PBS through x1
18. Resuspend pellet with 500uL buffer, and apply directly through column
    1. You do not have to collect the flow-through
19. Wash the column with 3 mL buffer, x2
20. Remove the column from the magnetic field, and place in collection tube. Elute targeted population by adding 5mL buffer and pushing through with supplied plunger. This is the astrocyte fraction.
     

Counting and Plating Astrocytes

1. Centrifuge eluted cells 300*rcf for 5 mins, at 4C
2. Resuspend in 1ml warmed serum-free astrocyte media for counting using hemocytometer
3. Calculate cell number and resuspension volume required for density of cells @ 100-150K/350uL astrocyte media
4. Place 350 uL of resuspended cells into each well to plate @ 100-150K per well
5. Supplement with 150uL fresh media 1^st day post plating (bringing total well volume to 500uL)
6. Completely chang media on  3^rd day post plating
7. Change ½ of well volume to fresh, warmed astrocyte media media every 3-4 days for maintenance

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