In vitro MV uptake assay

In vitro MV uptake assay protocol

Harvest of pulmonary-marginated vascular cells:

1. Inject C57/BL6 mice with 20ng of LPS i.v. via tail vein for 2hrs.
2. Sacrifice mice via anaesthetic overdose with i.p. pentobarbitone, check pedal reflex and confirm death by femoral artery exsanguination.
3. Carry out midline thoracotomy to expose chest cavity, clamp down rib cage to both sides and pull diaphragm down with haemostat clamp.
4. Inject 20 units of heparin into the right ventricle.
5. Perform tracheostomy and instil 20ml/kg of air with syringe to inflate the lungs.
6. Pass through a 4-0 curved-needle suture through the apex of heart to secure the heart placement with clear visualisation of both ventricles for subsequent cannulation.
7. Loop a 5-0 suture under the twist of pulmonary artery and aorta, throw loose knot.
8. Loop a 2-0 suture under apex of heart, throw loose knot.
9. Poke hole in the right ventricle with 25G needle and insert PVC tubing sized OD 0.61mm x ID 0.28mm into pulmonary artery (soften tubing end by pulling the end gently and ensure tubing is filled with perfusate).
10. Tighten the 5-0 suture around the cannulated pulmonary artery to secure the inflow cannulation.
11. Poke hole in the left ventricle with 19G needle & insert PVC tubing sized OD 0.86mm x OD 1.27mm into left ventricle, take care not to rupture the heart.
12. Tighten the 2-0 suture around apex of heart to secure the outflow cannulation.
13. Start perfusion with a syringe pump at 10ml/hr for 1min to observe outflow of blood into the collection tube (keep perfusate on ice throughout).
14. Increase perfusion to 50ml/hr for 12mins to collect 10ml of pulmonary vascular cells.
15. Centrifuge cells at 300 x g for 10mins.
16. Wash cell pellet twice in HBSS (+Ca^2+/Mg²⁺).
17. Resuspend cells in 0.5% human albumin solution (HAS)-HBSS supplemented with penicillin-streptomycin.
18. Stain cells using antibody panel listed in the table below and count cells by flow cytometry.
     

Generation of MVs in vitro:

1. Plate J774A.1 cells the day before in 60 x 15 mm petri dishes at seeding density 4 x 10⁶ cells.
2. Next day, remove media and rinse adherent cells 3 times with calcium-free PBS.
3. Add 3mM of ATP in PBS (+Ca^2+/Mg²⁺) onto cells for 30 min at 37°C.
4. Harvest supernatant, centrifuge at 300 x g for 10mins at 4°C to remove cells.
5. Collect supernatant in fresh tubes and centrifuge at 20,000 x g for 30min at 4°C to pellet MVs.
6. Discard supernatant, wash and re-suspend MV pellet in 0.5% HAS-HBSS buffer.
7. Stain MVs with 5mM of DiD (Life Technologies) in Diluent C (Sigma Aldrich), incubate 7 min in the dark at room temperature.
8. Wash MVs twice with 0.5% HAS-HBSS, centrifuge at 20,000 x g for 30 min at 4°C to pellet MVs.
9. Re-suspend MV pellet in 0.5% HAS-HBSS buffer, lyse with 1% Triton X-100 and determine DiD fluorescence in a plate reader with 635nm excitation and 680nm emission wavelengths.
10. Adjust MV concentration according to DiD fluorescence. For DiD fluorescence-MV concentration relationship see Figure 1D,  J Extracell Vesicles. 2020 Jan 5;9(1):1706708. doi: 10.1080/20013078.2019.1706708.

MV uptake assay:

1. Divide pulmonary vascular cells into Eppendorf tubes standardised to Ly6C^high monocyte count (50,000 cells per 500µl volume)
2. Add DiD labelled MVs to cells, typically at 100,000FU/ml (see Figure 6A, J Extracell Vesicles. 2020 Jan 5;9(1):1706708. doi: 10.1080/20013078.2019.1706708)
3. Cover tubes with aluminium foil, place on a rotating wheel and incubate at 37°C for up to 1hr.
4. Collect tubes and place on ice to cool immediately.
5. Centrifuge samples at 300 x g for 10mins to pellet cells and remove MVs
6. Wash cells twice with cold PBS.
7. Stain cells (See Table) for flow cytometric analysis of MV-uptake. Cell-associated DiD fluorescent is detected in the APC channel and quantified as mean fluorescence intensity.

Antibodies for flow cytometric analysis of perfusate cells

Buffers: 

1. Perfusion buffer (4% HAS, 1% penicillin-streptomycin, in HBSS)
2. Incubation buffer (0.5% HAS, 1% penicillin-streptomycin, in HBSS)

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