6. Add 1 mL of inoculum per well of the 6-well dish and incubate
at 37
C in a CO 2 incubator for 4 h (see Note 21).
7. Wash cells twice with pre-warmed PBS to remove extracellular
bacteria.
8. Replace media in wells with 2 mL of Macrophage Media per
well of a 6-well dish.
9. Maintain hMDMs in culture for the desired amount of time as
indicated by experimental design.
10. Wash hMDMs once with pre-warmed PBS.
11. Add 1 mL of Immune Cell Detachment Buffer per well and
incubate for 10–15 min at 37
C in a CO 2 incubator (see Note
6).
12. Pipette hMDMs and Immune Cell Detachment Buffer in the
well up and down to insure robust detachment, and transfer to
1.5-mL Eppendorf tubes.
13. Centrifuge hMDMs for 5–10 min at 350 Â g.
14. Resuspend cells in Flow Cytometry Buffer and count using
Trypan Blue on a hemocytometer (see Note 7).
15. Aliquot samples according to experimental design. In the event
of multicolor flow cytometry, it is essential to include compensation controls in order to correct for spectral overlap of fluorochromes and fluorescent proteins.
16. Organize samples according to their staining conditions
(unstained, compensation controls, full panel).
17. Remove supernatant from cells to be stained with viability dye
and keep cells that will not be stained on ice.
18. Resuspend cells to be stained in 100 μL of 1:1000 viability dye
diluted in PBS and incubate for 15–30 min at RT in the dark
(see Note 10).
19. Centrifuge cells for 5–10 min at 350 Â g.
20. Wash all samples twice with at least 1 mL of Flow Cytometry
Buffer.
21. Incubate samples with 5 μL FcX TruStain in 45 μL Flow
Cytometry Buffer on ice for 5–10 min (see Note 22).
22. While incubating, prepare antibody cocktail for immune cell
surface epitopes in Flow Cytometry Buffer for a total volume of
50 μL. For Biolegend α-HLA-DR-APC antibody, dilute 5 μL
antibody in 45 μL Flow Cytometry Buffer.
23. Add surface antibody cocktail to samples according to staining
conditions and pipette mix with FcBlock/cell solution. Incubate on ice in the dark for 20 min.
24. Centrifuge cells for 5–10 min at 350 Â g.
Flow Cytometry for Mycobacteria
265
at 37
C in a CO 2 incubator for 4 h (see Note 21).
7. Wash cells twice with pre-warmed PBS to remove extracellular
bacteria.
8. Replace media in wells with 2 mL of Macrophage Media per
well of a 6-well dish.
9. Maintain hMDMs in culture for the desired amount of time as
indicated by experimental design.
10. Wash hMDMs once with pre-warmed PBS.
11. Add 1 mL of Immune Cell Detachment Buffer per well and
incubate for 10–15 min at 37
C in a CO 2 incubator (see Note
6).
12. Pipette hMDMs and Immune Cell Detachment Buffer in the
well up and down to insure robust detachment, and transfer to
1.5-mL Eppendorf tubes.
13. Centrifuge hMDMs for 5–10 min at 350 Â g.
14. Resuspend cells in Flow Cytometry Buffer and count using
Trypan Blue on a hemocytometer (see Note 7).
15. Aliquot samples according to experimental design. In the event
of multicolor flow cytometry, it is essential to include compensation controls in order to correct for spectral overlap of fluorochromes and fluorescent proteins.
16. Organize samples according to their staining conditions
(unstained, compensation controls, full panel).
17. Remove supernatant from cells to be stained with viability dye
and keep cells that will not be stained on ice.
18. Resuspend cells to be stained in 100 μL of 1:1000 viability dye
diluted in PBS and incubate for 15–30 min at RT in the dark
(see Note 10).
19. Centrifuge cells for 5–10 min at 350 Â g.
20. Wash all samples twice with at least 1 mL of Flow Cytometry
Buffer.
21. Incubate samples with 5 μL FcX TruStain in 45 μL Flow
Cytometry Buffer on ice for 5–10 min (see Note 22).
22. While incubating, prepare antibody cocktail for immune cell
surface epitopes in Flow Cytometry Buffer for a total volume of
50 μL. For Biolegend α-HLA-DR-APC antibody, dilute 5 μL
antibody in 45 μL Flow Cytometry Buffer.
23. Add surface antibody cocktail to samples according to staining
conditions and pipette mix with FcBlock/cell solution. Incubate on ice in the dark for 20 min.
24. Centrifuge cells for 5–10 min at 350 Â g.
Flow Cytometry for Mycobacteria
265