Phagocytosis assays

Myelin phagocytosis assay

For Flow cytometry

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

2. Plate 100-200 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 48h. 

3. Change to fresh culture medium containing conjugated (Fluoromyelin, Cell vue, pHrodo) or unconjugated myelin or apoptotic cells and culture for 30 to 180 min.

4. Wash *2 in PBS. 

5. Wash out unbound LC3 using 100ul Digitonin (50-250ug/mL in PBS) 10 min at room temp. 

This is a crucial step. Conc. and time sometimes needs to be adjusted. 

6. Fill up with PBS, resuspend, spin down at 300g, invert. Repeat once again. 

Fixation and staining

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

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

9. 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). 

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

11. Stain the cells with LC3, antibodies and dyes targeting the phagocytosed myelin or cells (if added in unconjugated form).

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. 

14. Dilute in 50-100ul PBS. 

15. Read in cytometer 

For ICC

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

2. Plate 100-200 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 48h. 

3. Change to fresh culture medium containing conjugated (Fluoromyelin, Cell vue, pHrodo) or unconjugated myelin or apoptotic cells and culture for 30 to 180 min.

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

5. Wash out unbound LC3 using Digitonin (50-250ug/mL in PBS) 10 min at room temp. 

This is a crucial step. Conc. and time sometimes needs to be adjusted. 

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

Fix perm

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

8. 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

9. 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 

10. 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.

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

14. 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). 

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

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

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

18. Read in confocal microscope with plate adaptor. 

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. 

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