25. Wash all samples twice with at least 1 mL of Flow Cytometry
Buffer.
26. Remove supernatant and resuspend pellet in 4% paraformaldehyde for at least 15 min (see Note 23).
27. Wash fixed hMDMs twice with at least 1 mL of Flow Cytometry Buffer.
28. Resuspend fixed hMDMs in at least 250 μL of Flow Cytometry
Buffer (see Notes 24 and 25).
29. Transfer samples to round-bottom tubes and run flow cytometer according to manufacturer’s instructions (see
Note 26).
30. To perform doublet discrimination, be sure to acquire FSC-A
and FSC-H. To identify doublets, plot FSC-A versus FSC-H.
Events that are not on the diagonal of this plot are traditionally
considered doublets and excluded from downstream analysis
(Fig. 2, see Note 27).
31. Next, perform live-cell gating according to manufacturer’s
instructions for viability dye.
32. Acquire sufficient numbers of live, non-doublet events as indicated by experimental design.
33. Analyze resulting .fcs files using FlowJo or alternative flow
cytometry workflows (Fig. 2).
4 Notes
1. M. tuberculosis H37Rv and other mycobacterial strains can be
obtained from BEI Resources (Manassas, VA) or ATCC
(American Type Culture Collection) or other collections.
2. Alternative culture media for mycobacterial culture such as
Sauton’s media or a defined minimal medium can be used.
3. Alternative buffers compatible with flow cytometry protocols
such as Cell Staining Buffer from Biolegend or Stain Buffer
(FBS) from BD Biosciences can be used at this stage. Buffers
containing EDTA and/or detergent are helpful to minimize
cell clumping.
4. Other immune and non-immune cells infected with mycobacteria such as alveolar macrophages, dendritic cells, and neutrophils can be analyzed by flow cytometry.
5. Human macrophages can be differentiated from CD14+
monocytes using established protocols [7].
6. In the event of analysis of infected cells that are not human
macrophages, Macrophage Media should be replaced with the
appropriate culture media needed for maintenance of cells of
interest.
266
Sydney L. Solomon and Bryan D. Bryson
Buffer.
26. Remove supernatant and resuspend pellet in 4% paraformaldehyde for at least 15 min (see Note 23).
27. Wash fixed hMDMs twice with at least 1 mL of Flow Cytometry Buffer.
28. Resuspend fixed hMDMs in at least 250 μL of Flow Cytometry
Buffer (see Notes 24 and 25).
29. Transfer samples to round-bottom tubes and run flow cytometer according to manufacturer’s instructions (see
Note 26).
30. To perform doublet discrimination, be sure to acquire FSC-A
and FSC-H. To identify doublets, plot FSC-A versus FSC-H.
Events that are not on the diagonal of this plot are traditionally
considered doublets and excluded from downstream analysis
(Fig. 2, see Note 27).
31. Next, perform live-cell gating according to manufacturer’s
instructions for viability dye.
32. Acquire sufficient numbers of live, non-doublet events as indicated by experimental design.
33. Analyze resulting .fcs files using FlowJo or alternative flow
cytometry workflows (Fig. 2).
4 Notes
1. M. tuberculosis H37Rv and other mycobacterial strains can be
obtained from BEI Resources (Manassas, VA) or ATCC
(American Type Culture Collection) or other collections.
2. Alternative culture media for mycobacterial culture such as
Sauton’s media or a defined minimal medium can be used.
3. Alternative buffers compatible with flow cytometry protocols
such as Cell Staining Buffer from Biolegend or Stain Buffer
(FBS) from BD Biosciences can be used at this stage. Buffers
containing EDTA and/or detergent are helpful to minimize
cell clumping.
4. Other immune and non-immune cells infected with mycobacteria such as alveolar macrophages, dendritic cells, and neutrophils can be analyzed by flow cytometry.
5. Human macrophages can be differentiated from CD14+
monocytes using established protocols [7].
6. In the event of analysis of infected cells that are not human
macrophages, Macrophage Media should be replaced with the
appropriate culture media needed for maintenance of cells of
interest.
266
Sydney L. Solomon and Bryan D. Bryson