8. Amicon tubing and fittings (see Note 8).
9. 5 kDa MWCO ultrafiltration membrane (see Note 9).
10. Compressed N 2 cylinder.
11. Nitrogen regulator.
12. Magnetic stirrer.
13. 0.2μm polyethersulfone (PES) filter unit (see Note 10).
14. Wrench.
15. Teflon or polytetrafluoroethylene (PTFE) tape.
16. Collection containers.
17. SDS-PAGE gel.
18. Bicinchoninic acid (BCA) protein assay kit (see Note 11).
2.3 Enrichment
of Mycobacterium spp.
Extracellular Vesicles
1. Concentrated culture filtrate proteins (CFP) from Mycobacterium spp. (see Subheading 3.2, see Note 12).
2. 100 kDa MWCO centrifugal filter.
3. 3 kDa MWCO centrifugal filter.
4. Phosphate-buffered saline (PBS): 1 mM monopotassium phosphate (KH 2 PO 4 ) 0.14 g/L, 154 mM sodium chloride (NaCl)
8.99 g/L, and 5.6 mM disodium phosphate (Na 2 HPO 4 )
0.79 g/L in ultrapure water, adjusted to pH 7.4 and filtersterilized.
5. Capto™ Core 700 multimodal chromatography resin (see
Note 13).
6. Disposable gravity flow column with cap.
7. Leur-lock syringe.
8. 0.22μm polyethersulfone (PES) syringe filter.
9. Bicinchoninic acid (BCA) protein assay kit (see Note 11).
10. microBCA protein assay kit (see Note 14).
11. SDS-PAGE gel.
12. Nanoparticle Tracking Analysis (NTA) instrumentation (see
Note 15).
2.4 Fracturing
of Whole
Mycobacterium spp.
Cells by French Press
1. γ-Irradiated Mycobacterium spp. cells (see Subheading 3.1).
2. Phosphate-buffered saline (PBS) (see Subheading 2.3, item 4).
3. PBS-EDTA: PBS, pH 7.4 with 1 mM EDTA.
4. Protease inhibitor tablet, complete mini EDTA-free (Roche).
5. DNase: 1 mg/mL stock in water (stored at À20
C).
6. RNase: 1 mg/mL stock in water (stored at À20
C).
7. Breaking buffer: 50 mL PBS-EDTA, 30μL DNase (see item 5),
30μL RNase (see item 6), and protease inhibitor tablet (see item
4) (see Note 16).
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Megan Lucas et al.
9. 5 kDa MWCO ultrafiltration membrane (see Note 9).
10. Compressed N 2 cylinder.
11. Nitrogen regulator.
12. Magnetic stirrer.
13. 0.2μm polyethersulfone (PES) filter unit (see Note 10).
14. Wrench.
15. Teflon or polytetrafluoroethylene (PTFE) tape.
16. Collection containers.
17. SDS-PAGE gel.
18. Bicinchoninic acid (BCA) protein assay kit (see Note 11).
2.3 Enrichment
of Mycobacterium spp.
Extracellular Vesicles
1. Concentrated culture filtrate proteins (CFP) from Mycobacterium spp. (see Subheading 3.2, see Note 12).
2. 100 kDa MWCO centrifugal filter.
3. 3 kDa MWCO centrifugal filter.
4. Phosphate-buffered saline (PBS): 1 mM monopotassium phosphate (KH 2 PO 4 ) 0.14 g/L, 154 mM sodium chloride (NaCl)
8.99 g/L, and 5.6 mM disodium phosphate (Na 2 HPO 4 )
0.79 g/L in ultrapure water, adjusted to pH 7.4 and filtersterilized.
5. Capto™ Core 700 multimodal chromatography resin (see
Note 13).
6. Disposable gravity flow column with cap.
7. Leur-lock syringe.
8. 0.22μm polyethersulfone (PES) syringe filter.
9. Bicinchoninic acid (BCA) protein assay kit (see Note 11).
10. microBCA protein assay kit (see Note 14).
11. SDS-PAGE gel.
12. Nanoparticle Tracking Analysis (NTA) instrumentation (see
Note 15).
2.4 Fracturing
of Whole
Mycobacterium spp.
Cells by French Press
1. γ-Irradiated Mycobacterium spp. cells (see Subheading 3.1).
2. Phosphate-buffered saline (PBS) (see Subheading 2.3, item 4).
3. PBS-EDTA: PBS, pH 7.4 with 1 mM EDTA.
4. Protease inhibitor tablet, complete mini EDTA-free (Roche).
5. DNase: 1 mg/mL stock in water (stored at À20
C).
6. RNase: 1 mg/mL stock in water (stored at À20
C).
7. Breaking buffer: 50 mL PBS-EDTA, 30μL DNase (see item 5),
30μL RNase (see item 6), and protease inhibitor tablet (see item
4) (see Note 16).
80
Megan Lucas et al.