reactive oxygen species and pH. Flow, image, and mass cytometry
enable the quantification of tens of features simultaneously per cell
enabling high-dimensional characterization of population dynamics. Here, we describe two standard approaches for characterizing
mycobacteria via flow cytometry and characterizing macrophages
infected with M. tuberculosis. These protocols can be modified
according to experimental needs to accommodate measurement
of additional proteins, genes, and other biomolecules.
2 Materials
2.1 Flow Cytometry
of Mycobacteria
1. Mycobacterial strains (see Note 1).
2. 7H9 Medium: dissolve 4.7 g Middlebrook 7H9 powder
(Difco) in 900 mL dH 2 O, add 100 mL of 10% (v/v) oleic
acid, albumin, dextrose, catalase (OADC) supplement (Becton
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).
3. Spectrophotometer.
4. 15-mL conical tubes.
5. Fixation Buffer: 1Â PBS, 4% Paraformaldehyde (v/v). Store at
4
C.
6. 5-μm syringe filters.
7. 5- to 10-mL syringe.
8. 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).
9. 12 Â 75 mm, 5-mL polystyrene round-bottom tubes.
10. BD Fortessa Flow Cytometer.
11. FlowJo v10.
2.2 Flow Cytometry
of
Mycobacteria-Infected
Immune Cells
1. Human monocyte-derived macrophages (hMDMs) plated in a
6-well dish with 2 mL of Macrophage Media per well (see
Notes 4 and 5).
2. Macrophage Media: 1Â RPMI 1640, 10% (v/v) heatinactivated FBS, 2 mM L-glutamine, 10 mM HEPES. Filter
through a 0.22-μm Corning filter. Store at 4
C. Pre-warm to
37
C before use (see Note 6).
3. Fluorescent mycobacterial strain.
4. 7H9 Medium: dissolve 4.7 g Middlebrook 7H9 powder
(Difco) in 900 mL dH 2 O, add 100 mL of 10% (v/v) oleic
acid, albumen, dextrose, catalase (OADC) supplement (Becton
262
Sydney L. Solomon and Bryan D. Bryson
enable the quantification of tens of features simultaneously per cell
enabling high-dimensional characterization of population dynamics. Here, we describe two standard approaches for characterizing
mycobacteria via flow cytometry and characterizing macrophages
infected with M. tuberculosis. These protocols can be modified
according to experimental needs to accommodate measurement
of additional proteins, genes, and other biomolecules.
2 Materials
2.1 Flow Cytometry
of Mycobacteria
1. Mycobacterial strains (see Note 1).
2. 7H9 Medium: dissolve 4.7 g Middlebrook 7H9 powder
(Difco) in 900 mL dH 2 O, add 100 mL of 10% (v/v) oleic
acid, albumin, dextrose, catalase (OADC) supplement (Becton
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).
3. Spectrophotometer.
4. 15-mL conical tubes.
5. Fixation Buffer: 1Â PBS, 4% Paraformaldehyde (v/v). Store at
4
C.
6. 5-μm syringe filters.
7. 5- to 10-mL syringe.
8. 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).
9. 12 Â 75 mm, 5-mL polystyrene round-bottom tubes.
10. BD Fortessa Flow Cytometer.
11. FlowJo v10.
2.2 Flow Cytometry
of
Mycobacteria-Infected
Immune Cells
1. Human monocyte-derived macrophages (hMDMs) plated in a
6-well dish with 2 mL of Macrophage Media per well (see
Notes 4 and 5).
2. Macrophage Media: 1Â RPMI 1640, 10% (v/v) heatinactivated FBS, 2 mM L-glutamine, 10 mM HEPES. Filter
through a 0.22-μm Corning filter. Store at 4
C. Pre-warm to
37
C before use (see Note 6).
3. Fluorescent mycobacterial strain.
4. 7H9 Medium: dissolve 4.7 g Middlebrook 7H9 powder
(Difco) in 900 mL dH 2 O, add 100 mL of 10% (v/v) oleic
acid, albumen, dextrose, catalase (OADC) supplement (Becton
262
Sydney L. Solomon and Bryan D. Bryson