Myelin isolation and staining

Myelin isolation and purification, Rasmus Berglund 2016

Myelin isolation

1. Mince brain (2-4) and homogenize with glass plunger.
2. Apply to a 38% Percoll gradient 
3. Collect the myelin sheeth with 10 or 25mL pipette. 
4. Mix the myelin layer with 20mL 0.3M Sucrose in a 50mL Falcon tube. 
5. Underlay 20mL 0.83M sucrose. Dont mix the layers. 
6. Centrifuge at 75.000G for 30 minutes in 4C. 
7. Collect the myelin debris from the interface of the two sucrose densities.
8. Mix the myelin with 18 mL 20 mM Tris-Cl. Pipet up and down to homogenize
9. Divide in two 15mL Falcon tubes and centrifuge for 20 minutes at 12.000G 
10. Collect the purified white myelin pellet. Wash in ddH20. Label and/or keep at -80. (Conc. can be quantified with BSA kits etc.)
11. Label with PhRodo (Invitrogen), CellVue (eBioscience), PKH26 (Sigma Aldrich) according to instruction provided from manufacture.  

Solutions

20mL Tris-Cl
800mL distilled deionized H2O (ddH2O) add 20 mL of 1 M Tris·Cl, pH 7.45 (20 mM final concentration) and 20 mL of 100 mM Na2EDTA (2 mM final concentration). Adjust pH to 7.45. Adjust volume to 1000 mL with ddH2O. Filter solution through a 0.2 µm filtration unit.

Sucrose
Prepare 1 M sucrose solution. 
To 100 mL of Tris-Cl buffer solution, add 68.46 g of sucrose. Adjust volume to 200 mL with Tris-Cl buffer solution. Filter solution through a 0.2 µm filtration unit. Dilute with Tris-Cl buffer solution to prepare 0.32 M and 0.83 M sucrose solutions. 

Myelin phagocytosis assays

Flow cytometry

1. For Flow cytometry
2. Sort microglia from MOG + CFA immunized mice (day 5 p.i) CNS by MACS or FACS.
3. Plate 1-5*10^4 cells in culture medium per well in Poly-L-lysine coated (1 hour, 3 washes, dry) flat-bottom 96 well plates. Let the cells attach for 24h.
4. Change to fresh culture medium containing conjugated (Fluoromyelin, Cell vue, pHrodo) or unconjugated myelin or apoptotic cells and culture for 30 to 180 min.
5. Wash *3 by slowly adding MACS buffer and invert the plate. 

Fixation and staining

1. Detach cells with EDTA (2-5mM, 30-60 min, pipett up and down to detach cells). Transfer to V-bottom plate.

1. If surface molecules are to be detected. Add 50uL ab mixture and incubate in +4 for 20 min.

1. Wash twice by resuspending in MACS buffer, spin down at 300g and invert the plate.

1. Add Fix/Perm BD Fixation/Permeabilization buffer (100ul/well) incubate at +4⁰C for 20-30min. 

1. Add 150ul of cold BD Perm/Wash solution (diluted 10x from stock in distilled water) to all wells and spin down at 400g (after fixation cells need higher g force during centrifugation to be pelleted). 

1. Fill up wells with BD perm/wash, resuspend, spin down at 400g, invert. Repeat once again.

1. Stain the cells with antibodies and dyes targeting the phagocytosed myelin or cells (if added in unconjugated form).

1. Add antibody mixture in 50ul/well to all wells. Pre-dilute antibodies in cold BD Perm/Wash solution so all samples get exactly the same amount of antibody. Pipette all wells thoroughly and incubate for 30 minutes at 4⁰C.

1. Fill up wells with BD perm/wash, resuspend, spin down at 400g, invert. Repeat once again. Dilute in 50-200ul PBS.

1. Read in cytometer 
   1. Sort microglia from MOG + CFA immunized mice CNS by MACS or FACS. 

ICC

1. Plate 1-2*10^4 cells in culture medium per well in Poly-L-lysine coated (1 hour, 3 washes, dry) flat bottom 96 well plates (make sure it fits your confocal microscope adaptor).  Let the cells attach for 24h. 
2. Change to fresh culture medium containing conjugated (Fluoromyelin, Cell vue, pHrodo) or unconjugated myelin or apoptotic cells and culture for 30 to 180 min.
3. Wash *2 by slowly pouring PBS to the wells and the invert. 
4. Wash *2 by slowly pouring PBS to the wells and the invert. 

Fixation and permeabilization

1. Add PFA (100ul/well)  and incubate 10 min at room temp. Wash *2 by slowly pouring PBS to the wells and the invert. 
2. Add Tween-20 0,2% in PBS (100ul/well) and incubate 15 min at room temp. Wash *2 by slowly pouring PBS to the wells and the invert.

Blocking and staining

1. Blocking: Add w/o washing step PBS+0.2% Tween-20 and 2% BSA and 20% of serum from secondary antibody producing species. Incubate for 40m 
2. Incubate cells with diluted primary ab 0.1 ul (diluted 1:1000, 100 ul per well) per well. in PBS+0.2%Tween20 + 1% serum from antibody host (ex goat) and 1% BSA for in 4+ overnight. If you use conjugated antibody, use 1% BSA instead of host serum.
3. Wash *2 by slowly pouring PBS to the wells and the invert. 
4. Incubate cells with diluted secondary antibody 1:200. 1 ul per well diluted in 50ul PBS+0.2%Tween-20 +1% serum from ab host 1h at room temp /in the dark). 
5. Wash *2 by slowly pouring PBS to the wells and the invert. 
6. Add DAPI solution for 1 minute, then wash 3 times. 
7. Fill all wells with PBS and cover with plastic and aluminumfoil. Keep max 2 weeks before reading. 
8. Read in confocal microscope with plate adaptor. 

Myelin clearance assay

1. Sort microglia from MOG + CFA immunized mice (5 days p.i) CNS by MACS or FACS. 
2. Seed cells in flat bottom 96 well plates, 2-5*10^4 cells per well. 
3. Incubate 12-24 hours in complete medium. 
4. Aim for an even 80-90% cell confluency. Discard unattached cells. 
5. Pulse cells with PKH26 labeled myelin for 30-60 min in complete DMEM. 
6. Aspirate the supernatant and place in e.g, 2mL tubes.
7. Add PBS, spin 300g for 1 min 
8. Place supernatant in 96 well plate suitable for your plate reader. 
9. 5. Repeat 5-7 for 1-4 times. 
10. 6. Apply a dilution series with PKH26 to the plate to use as reference curve for calculating arbitrary units. 
11. Read the plate in a plate reader. We used a SpectraMax 384 microplate reader for fluorescence at 560nm. If focal plane is applied adjust to peak intensity. The remaining myelin concentration was determined in relation to a standard dilution series.

Prior to experiments the myelin concentration needs miticulus concentration in relation to number of cells per well. In our experience the interval is small and needs to be titrated per myelin batch. 

Culture medium: DMEM High glucose + 10ml MM + 5ml PenStrep (Sigma P4458) + 10 % FCS + 10ng/ml M-CSF (R&D 416-ML-050)

MM – Magic Mix preparation: 100mL L-Glutamine, 100mL Sodium pyruvate, 4,5 mL 2-mercaptoethanol.

Protocol by Rasmus Berglund, MD PhD Karolinska institute. Contact rasmus.berglund@ki.se for further details.