16. It is advisable to consider the autofluorescence properties of
the immune cells being analyzed. In the event of autofluorescence, it is beneficial to select a fluorescent mycobacterial strain
that has the least amount of interference with the immune cell’s
autofluorescence.
17. Alternative protocols for infection are possible. For example,
one can use mycobacteria from a different phase of growth
(stationary phase vs. log phase). Furthermore, it is preferred
to wash away 7H9 M. tuberculosis Growth Media prior to
infection; however, if desired, this step can be omitted, and
mycobacterial optical density can be measured and media containing mycobacteria for infection can be prepared by mixing
Macrophage Media with the appropriate culture volume of
H37Rv M. tuberculosis.
18. Instead of using fluorescent mycobacteria to identify infected
populations of immune cells, it is possible to perform intracellular staining using fluorescent antibodies that recognize
M. tuberculosis or M. tuberculosis proteins. If this approach is
desirable, following infection and detachment of immune cells
and staining with a fixation-compatible viability dye (step 13 of
Subheading 3.2), infected immune cells are fixed. Following
fixation, fixed cells are centrifuged and then permeabilized.
Following permeabilization, fixed cells are stained with fluorescent antibodies that recognize M. tuberculosis or M. tuberculosis
proteins. After incubating with fluorescent antibodies, proceed
with step 16 of Subheading 3.2.
19. Another method to generate a disperse suspension of mycobacteria following centrifugation involves 2–3 rounds of brief
sonication.
20. A useful approximation to convert between OD 600 and Mtb
concentration is that an OD 600 ¼ 1 is approximately equal to
3 Â 10
8 CFU/mL.
21. Alternative protocols for infection are possible. For example, if
monitoring the dynamics of phagocytosis, it is preferred to
sample several different time points following infection. It is
also possible to further alter the volume of Macrophage Media
used during infection. Infecting in a reduced culture volume
enhances M. tuberculosis uptake. In order to measure phagocytosis rates, it is good practice to use at least one well as a sham
infection control where media is changed but no
M. tuberculosis is added.
22. It is important to consider whether blocking of Fc receptors on
the surface of your immune cells is important. If blocking of Fc
is important, perform Fc blocking prior to staining with cells
with fluorescent antibodies recognizing surface proteins of
interest.
Flow Cytometry for Mycobacteria
269
the immune cells being analyzed. In the event of autofluorescence, it is beneficial to select a fluorescent mycobacterial strain
that has the least amount of interference with the immune cell’s
autofluorescence.
17. Alternative protocols for infection are possible. For example,
one can use mycobacteria from a different phase of growth
(stationary phase vs. log phase). Furthermore, it is preferred
to wash away 7H9 M. tuberculosis Growth Media prior to
infection; however, if desired, this step can be omitted, and
mycobacterial optical density can be measured and media containing mycobacteria for infection can be prepared by mixing
Macrophage Media with the appropriate culture volume of
H37Rv M. tuberculosis.
18. Instead of using fluorescent mycobacteria to identify infected
populations of immune cells, it is possible to perform intracellular staining using fluorescent antibodies that recognize
M. tuberculosis or M. tuberculosis proteins. If this approach is
desirable, following infection and detachment of immune cells
and staining with a fixation-compatible viability dye (step 13 of
Subheading 3.2), infected immune cells are fixed. Following
fixation, fixed cells are centrifuged and then permeabilized.
Following permeabilization, fixed cells are stained with fluorescent antibodies that recognize M. tuberculosis or M. tuberculosis
proteins. After incubating with fluorescent antibodies, proceed
with step 16 of Subheading 3.2.
19. Another method to generate a disperse suspension of mycobacteria following centrifugation involves 2–3 rounds of brief
sonication.
20. A useful approximation to convert between OD 600 and Mtb
concentration is that an OD 600 ¼ 1 is approximately equal to
3 Â 10
8 CFU/mL.
21. Alternative protocols for infection are possible. For example, if
monitoring the dynamics of phagocytosis, it is preferred to
sample several different time points following infection. It is
also possible to further alter the volume of Macrophage Media
used during infection. Infecting in a reduced culture volume
enhances M. tuberculosis uptake. In order to measure phagocytosis rates, it is good practice to use at least one well as a sham
infection control where media is changed but no
M. tuberculosis is added.
22. It is important to consider whether blocking of Fc receptors on
the surface of your immune cells is important. If blocking of Fc
is important, perform Fc blocking prior to staining with cells
with fluorescent antibodies recognizing surface proteins of
interest.
Flow Cytometry for Mycobacteria
269