Dickinson), 4 mL of 50% (v/v) glycerol, 5 mL of 10% (v/v)
Tween-80. Filter through a 0.22-μm Corning filter. Store at
4
C (see Note 2).
5. 15-mL conical tubes.
6. 5-μm syringe filters.
7. 5- to 10-mL syringe.
8. Spectrophotometer.
9. PBS.
10. Immune Cell Detachment Buffer: 1Â PBS, 2 mM EDTA.
Filter through a 0.22-μm Corning filter. Store at 4
C.
Pre-warm to 37
C before use (see Note 7).
11. Flow Cytometry Buffer: 1Â PBS, 2% (v/v) heat-inactivated
FBS, 2 mM EDTA. Filter through a 0.22-μm Corning filter.
Store at 4
C (see Note 3).
12. Trypan Blue.
13. Disposable hemocytometer (see Note 8).
14. 1.5-mL clear Eppendorf tubes (see Note 9).
15. Flow Cytometry-compatible Cell Viability Dye (ex: fixable
Zombie dyes provided by Biolegend resuspended at 1:1000
in 1Â PBS) (see Note 10).
16. Human TruStain FcX (Biolegend).
17. Human anti-HLA-DR antibody conjugated to APC (Biolegend, clone L243).
18. Fixation Buffer: 1Â PBS, 4% (v/v) Paraformaldehyde. Store at
4
C.
19. 12 Â 75 mm, 5-mL polystyrene round-bottom tubes.
20. BD Fortessa.
21. FlowJo v10.
3 Methods
3.1 Flow Cytometry
of Mycobacteria
1. Culture mycobacterial strain in 7H9 medium. Measure growth
by OD 600 . Upon achieving experimental desired optical density, prepare samples for flow cytometry.
2. Transfer appropriate culture volume to 15-mL conical tubes
and centrifuge for at least 5 min at 3000 Â g (see Note 11).
3. Resuspend mycobacterial pellet in 5–10 mL of Fixation Buffer
and incubate for at least 15 min (or longer if needed for BSL3
inactivation protocols) (see Notes 12–14).
Flow Cytometry for Mycobacteria
263
Tween-80. Filter through a 0.22-μm Corning filter. Store at
4
C (see Note 2).
5. 15-mL conical tubes.
6. 5-μm syringe filters.
7. 5- to 10-mL syringe.
8. Spectrophotometer.
9. PBS.
10. Immune Cell Detachment Buffer: 1Â PBS, 2 mM EDTA.
Filter through a 0.22-μm Corning filter. Store at 4
C.
Pre-warm to 37
C before use (see Note 7).
11. Flow Cytometry Buffer: 1Â PBS, 2% (v/v) heat-inactivated
FBS, 2 mM EDTA. Filter through a 0.22-μm Corning filter.
Store at 4
C (see Note 3).
12. Trypan Blue.
13. Disposable hemocytometer (see Note 8).
14. 1.5-mL clear Eppendorf tubes (see Note 9).
15. Flow Cytometry-compatible Cell Viability Dye (ex: fixable
Zombie dyes provided by Biolegend resuspended at 1:1000
in 1Â PBS) (see Note 10).
16. Human TruStain FcX (Biolegend).
17. Human anti-HLA-DR antibody conjugated to APC (Biolegend, clone L243).
18. Fixation Buffer: 1Â PBS, 4% (v/v) Paraformaldehyde. Store at
4
C.
19. 12 Â 75 mm, 5-mL polystyrene round-bottom tubes.
20. BD Fortessa.
21. FlowJo v10.
3 Methods
3.1 Flow Cytometry
of Mycobacteria
1. Culture mycobacterial strain in 7H9 medium. Measure growth
by OD 600 . Upon achieving experimental desired optical density, prepare samples for flow cytometry.
2. Transfer appropriate culture volume to 15-mL conical tubes
and centrifuge for at least 5 min at 3000 Â g (see Note 11).
3. Resuspend mycobacterial pellet in 5–10 mL of Fixation Buffer
and incubate for at least 15 min (or longer if needed for BSL3
inactivation protocols) (see Notes 12–14).
Flow Cytometry for Mycobacteria
263