Myelin phagocytosis and clearance assays

Myelin isolation, purification and myelin phagocytosis assays

Rasmus Berglund KI 2016

Myelin isolation

1.                   Mince brain and homogenize with glass plunger. 

2.                   Apply to a 38% Percoll gradient 

3.                   Mix the myelin layer with 20mL 0.3M Sucrose in a 50mL Falcon tube. 

4.                   Underlay 20mL 0.83M sucrose. Dont mix the layers. 

5.                   Centrifuge at 75.000G for 30 minutes in 4C. 

6.                   Collect the myelin debris from the interface of the two sucrose densities.

7.                   Mix the myelin with 18 mL 20 mM Tris-Cl. Pipet up and down to homogenize

8.                   Divide in two 15mL Falcon tubes and centrifuge for 20 minutes at 12.000G 

9.                   Collect the purified white myelin pellet. Wash in ddH₂0. Label and/or keep at-80. Conc. can be quantified with BSA kits etc followed by titration for specific assays witch is key to successful experiments. 

10.                 Conjugation to PKH26, pHRhodo, Cellvue according to manufacteurs instructions. Other lipophilic dyes can likely also be used.

20 mM 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. 

Dyes

PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling Cat. no PKH26GL. Sigma-Aldrich 

CellVue™ Plum Cell Labeling Kit, Cat. no: 88-0871-16 Invitrogen

pHrodo™ Red, succinimidyl ester (pHrodo™ Red, SE), Cat. no: P36600, Invitrogen

pHrodo™ Red and Green AM Intracellular pH Indicator Dyes, Cat. no: P35373, Invitrogen

Microglial myelin phagocytosis assays

This protocol is developed for analysis of myelin phagocytosis by microglia and other CNS macrophages but can likely be used for other cells and phagocytosed compounds. 

Flow cytometry

1.                   Sort microglia from MOG + CFA immunized mice (day 5 p.i) CNS by MACS (CD11b, 130-093-636, Miltenyi                           Biotec) or FACS (various gating strategies, e.g, Live – CD11b+ Ly6G- CD45^Intermediate ). If sorted later that                           day 5 CD11b MACS sorting can’t be considered microglia specific due to infiltration of of peripheral immune                         cells.

2.                   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 (PKH26, 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 intracellular staining (if targeting intracellular proteins, otherwise read directly in cytometer)

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

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

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

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

10.                 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). 

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

12.                 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.

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

14.                 Read in cytometer 

ICC

1.                   Prepare single cell suspensions from mouse CNS 

2.                   Sort microglia from MOG + CFA immunized mice (day 5 p.i) CNS by MACS (CD11b, 130-093-636, Miltenyi                           Biotec) or FACS (various gating strategies, e.g, Live – CD11b+ Ly6G- CD45^Intermediate ). If sorted later that                           day 5 CD11b MACS sorting can’t be considered microglia specific due to infiltration of of peripheral immune                         cells.

3.                   Plate 1-2*10^4 cells in culture medium per well in Poly-L-lysine coated (1 hour,

                      3 washes in ddH₂O water, dry) flat bottom 96 well plates (make sure it fits your confocal microscope                                     adaptor). Let the cells attach for 24h. 

4.                   Change to fresh culture medium containing conjugated (PKH26, Cellvue, pHrodo) or unconjugated myelin or                       apoptotic cells and culture for 30 to 180 min. For comprehensive analysis, use a pH sensitive dye e.g.,                                  pHrodo together with a pH-indifferent dye to detect both phagocytosed and lysosome-loaded myelin.  

5.                   Wash *2 by slowly pouring PBS to the wells and the invert. 

Fixation and permeabilization

6.                   Add PFA (100ul/well)  and incubate 10 min at room temp. Wash *2 by slowly pouring PBS to the wells and                             invert the plate. 

7.                   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 the plate.

Blocking and staining

8.                   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. 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. 

                      If myelin were added unconjugated: Stain the cells with antibodies (e.g, anti-MOG Merck MAB5680, anti-                             dMBP, Merck AB5864  ) or dyes targeting the phagocytosed myelin or cells e.g, Fluoromyelin™.

9.                   Wash *2 by slowly pouring PBS to the wells and invert the plate. 

10.                 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). 

11.                 Wash *2 by slowly pouring PBS to the wells and the invert. 

12.                 Add DAPI solution for 1 minute, then wash 3 times. 

13.                 Fill all wells with PBS and cover with plastic and aluminumfoil. Keep max 2 weeks before reading. 

14.                 Read in confocal microscope with plate adaptor. 

Myelin clearance assay

1.                   Prepare single cell suspension from mouse CNS          

2.                   Sort microglia from MOG + CFA immunized mice (day 5 p.i) CNS by MACS (CD11b, 130-093-636, Miltenyi                           Biotec) or FACS (various gating strategies, e.g, Live – CD11b+ Ly6G- CD45^Intermediate ). If sorted later that                            day 5 CD11b MACS sorting can’t be considered microglia specific due to infiltration of of peripheral immune                          cells.

3.                   Seed cells in flat bottom 96 well plates, 2-5*10^4 cells per well. 

                       DMEM

4.                   Aim for an even 80-90% cell confluency. Discard unattached cells by aspirating medium and wash once by                           slowly pouring DMEM into wells and invert the plate. 

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 and replace medium with complete DMEM w/o myelin.

7.                   Add PBS, spin 300g for 1 min 

8.                   Place supernatant in 96 well plate suitable for your plate reader. 

9.                 Repeat 6-9 for 1-4 times with 1h intervals. 

10.                 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 meticulous concentration titration 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 Institutet. Do not hesitate to contact rasmus.berglund@ki.se 

for further details. 

Cite: 

Berglund R, Guerreiro-Cacais AO, Adzemovic MZ, et al. Microglial autophagy-associated phagocytosis is essential for recovery from neuroinflammation. Sci Immunol. Oct 16 2020;5(52)doi:10.1126/sciimmunol.abb5077