RNA isolation and qPCR

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 (to flame polish cover glass)
• Centrifuge (capable of 300 rcf, cooling to 4°C, and holding 15/50 mL tubes)
• 37°C Water bath shaker (optional, swirling every 5 minutes in stationary bath just as effective)
• Carbogen bubblers (Meduna’s Mixture, 95% O2: 5% CO2)
• Brain dissection equipment (ice stage, petri dish, forceps, surgical scissors)
• 15ml and 50ml conical tubes
• Pipettes (various sizes), pipette aid, serological pipettes (various sizes), fire-polished Pasteur pipettes
• 0.2 um sterile syringe filter and syringe
• 70 um sterile cell strainer
• Hemocytometer
• Mouse pups (P3 optimal, up to P5 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)
• Neuron dissection media
  Neuron Dissection Media (500mL):
  • 10mL 100mM Sodium Pyruvate
  • 500uL 20% glucose in ddH20 (sterile filtered)
  • 1.2g HEPES
  • 400mL HBSS
  • Mix until solutes dissolve and bring up to 500mL
  • Sterile filter in hood with Nalgene Rapid-Flow filter unit (0.2um pore, ThermoFisher 566-0020)
• 0.5% bovine serum albumin in phostphate buffered solution (0.5% BSA/PBS; 290-300 osmol)
  0.5% BSA in 1x PBS (500mL):
  • 2.5g BSA
  • 800mL of 1x PBS
  • Mix until solutes dissolve and bring up to 500mL
  • Sterile filter in hood with Nalgene Rapid-Flow filter unit (0.2um pore, ThermoFisher 566-0020)
• LS Columns (Miltenyi biotec, 130-042-401)
• Anti-CD11b+ microbeads (Miltenyi biotec, 130-093-634)
• Anti-myelin microbeads (Miltenyi biotec, 130-096-733)
• Anti-ACSA-2 microbead kit (Miltenyi biotec, 130-097-678)
• Serum-free astrocyte media
  Serum-free astrocyte media (125mL)
  • 60mL MEM (Thermo Fisher, 51200038)
  • 60mL NBM (Thermo Scientific, 21103049)
  • 2.5mL 50x Serum-free B27 (Thermo Fisher, 17504001)
  • 1.25mL 100mM Sodium Pyruvate (Thermo Scientific, 11360070)
  • 1.25mL 100x GlutaMAX Supplement (Thermo Fisher, 35050061)
  • 125uL Penicillin-Streptomycin (5000U/mL, Thermo Scientific, 15070063)
  • Sterile filter in hood with Nalgene Rapid-Flow filter unit (0.2um pore, ThermoFisher 566-0020)

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 for 2-24hr room temperature
• Wash coverslips with sterile ddH20 3 times
  • 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 coated plates can be stored at -20°C up to 1 month
• Place serum-free astrocyte media in 37°C 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. 
   1. Place albumin-ovomucoid inhibitor bottle on ice for later use
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. 
   1. I add DNase using 200 pipet at 125ulx2 (1000 tips don’t fit well in tiny bottle)
   2. Place bottle with remaining DNase on ice for later use
4. Vigorously bubble the Neuron Dissection Media with O2:CO2 on ice for 10-15 minutes prior to dissection
5. Bubble papain with O2:CO2 at room temperature alongside Dissection Media, with airflow slightly indenting surface rather than bubbling the mix vigorously
6. Remove Dissection Media from bubbling and place in petri dishes set in ice bucket
   1. Continue bubbling papain/DNase mix at room temperature
7. Dissect brain region of interest in petri dishes, then mince tissue into ~1-2mm pieces
   1. Typically, 3-4 pups will suffice for 18-24 wells of a 24 well-plate at 150K density
   2. Tissue can be dissected separately and combined during remainder of isolation process
8. Prepare the papain/DNAse mix by removing it from bubbling, then draw the solution into a syringe and expelling the contents through a 0.2um filter into a 50mL conical tube
9. Draw the minced tissue using a serological pipette, let the tissue separate from the dissection media, and then expel the settled tissue into the papain/DNase solution tube
10. Incubate the tissue and papain mix in the 37°C bath for 10 minutes, swirling mix every 5 minutes to maximize papain exposure
11. After the incubation period, titrate the mixture with serological pipette gently with pipette setting turned to slow-medium speed for 3-5 times (up and down cycle count as 1)
12. Centrifuge the homogenized solution at 300*rcf for 5 min at 4°C. 
13. 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. in a separate 50mL conical tube, add 5 mL of the albumin (vial 4)
14. Discard the supernatant and immediately resuspend the cell pellet in 1mL resuspension media
    1. Titrate on slow-medium, 5-7 times using fire-polished glass pipet to further homogenize tissue
    2. Add remaining volume of resuspension media to mixture after titration
15. Carefully layer the resuspended mixture on top of albumin, then centrifuge at 300*rcf for 5 min at 4°C. 
    1. During centrifuge, prepare a 50mL conical tube with a 70μm BD Falcon filter by adding 1mL 0.5% BSA in PBS to wet the filter
16. Discard the supernatant and immediately suspend the pelleted cells in 9 mL 0.5% BSA in PBS (referred to as buffer). Filter the solution using 70μm BD Falcon filter to remove any non-dissociated tissue.

Antibody-Microbead pull down

1. Centrifuge solution at 300*rcf for 5 min at 4°C; discard supernatant
2. Resuspend pellet in 150uL buffer
3. Add 15-20 uL Anti-CD11b+ microbeads
4. Add 15-20 uL Anti-myelin microbeads
5. Incubate 10 mins at 4°C (or ice bucket), mixing every 5 minutes
6. “Wash” by adding 1mL 0.5% BSA/PBS (“buffer”) to mixture
7. Centrifuge at 300*rcf for 3 mins at 4°C, discard supernatant
   1. This will remove any excess beads from solution
   2. While centrifuging, prep for magnetic separation
      1. Put LS column in holder with 50mL conical tube underneath, wash 2mL buffer through once
8. Resuspend pellet with 500uL buffer, and apply directly through column, collecting the flow through in the 50mL conical tube placed underneath (the fraction of cells that were not labeled, contains astrocytes + neurons)
9. Wash the column with 3mL buffer, 2 times
   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 4°C, discard supernatant
11. Resuspend pellet in 150uL buffer
12. Add 15-20uL of FcR blocking reagent from the Anti-ACSA-2 microbead kit
13. Incubate 10 minutes at 4°C (or on ice), mixing every 5 minutes
14. Add 15-20uL of ACSA-2 microbeads from the Anti-ACSA-2 microbead kit
15. Incubate 10 minutes at 4°C, mixing every 5 minutes
16. Wash by adding 1mL buffer to mixture
17. Centrifuge at 300*rcf for 3 mins at 4°C, 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 once
       2. Flow through can be collected in waste bucket if not needed or in 50mL conical tube if one is interested in keeping/using the flow through (mostly neurons and oddball cell types)
18. Resuspend pellet with 500uL buffer, and apply directly through column
19. Wash the column with 3 mL buffer, 2 times
20. Remove the column from the magnetic field, and place in collection tube. Elute targeted population by placing column in 15mL conical tube, then adding 5mL buffer to column and pushing through with supplied plunger.
    1. This is the astrocyte fraction
    2. Cell density can be calculated directly from this sample with hemocytometer methods, where the count is entered into a template spreadsheet (Cell Culture Calculation Template)
       1. See below for calculating cell number using alternative method

Counting and Plating Astrocytes

1. Centrifuge eluted cells 300*rcf for 5 mins, at 4°C
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
   1. (total count ÷ grids counted) x total initial sample volume [1mL] x 10⁴ = Total cell count
   2. Total cell count ÷ desired plating density [100,000 to 150,000] = Total wells possible
   3. Total wells possible x desired plating volume [350uL] = final total volume needed for sample
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 change media on  3^rd day post plating
7. Change ½ of well volume to fresh, warmed astrocyte media every 3-4 days for maintenance