23. In the event that there is access to a flow cytometry compatible
with live mycobacterial cultures, fixation is optional. In the
event that fixation is optional, infected cells can be directly
resuspended in Flow Cytometry Buffer. If performing livecell analysis to maintain cells in culture after cell sorting, do
not use Fixation Buffer. Instead, resuspend cells directly in
Flow Cytometry Buffer and perform experiments.
24. It is also possible to monitor intracellular protein expression in
immune cells by flow cytometry. If this is desired, following
infection, detachment of immune cells, staining with a fixationcompatible viability dye, and surface antibody staining if
desired (through step 19 of Subheading 3.2), infected
immune cells are fixed using Fixation Buffer. Following fixation, fixed cells are permeabilized and then stained with fluorescent antibodies that recognize host proteins of interest.
After incubating with fluorescent antibodies, proceed from
step 20 as described in Subheading 3.2.
25. It is also possible to monitor additional features of the hostpathogen interaction using commercially available dyes
(ex. LysoSensor for pH, MitoSox for metabolic state)
[12, 13]. If this is desirable, the above protocol may need to
be modified in order to accommodate manufacturer-specific
protocols.
26. It is possible to analyze these samples using other techniques
such as mass cytometry or image cytometry. Mass cytometry
enables analysis of tens of proteins simultaneously on the same
cell; however, fluorescent mycobacteria cannot be identified
using this technique. Image cytometry, though lower throughput and fewer channels available for interrogation, enables the
analysis of protein localization in addition to expression
[4, 14].
27. FSC and SSC should be acquired on a linear scale to discriminate cells from debris. Alternative methods for doublet discrimination
are
available
including
acquiring
FSC-A vs. FSC-W, FSC-H vs. FSC-W, or SSC-A vs. SSC-W
among others.
Acknowledgments
This work was supported by the Ferry Fund (MIT), the Terry and
Susan Ragon Foundation, The Ragon Institute, and the MIT
Department of Biological Engineering.
270
Sydney L. Solomon and Bryan D. Bryson
with live mycobacterial cultures, fixation is optional. In the
event that fixation is optional, infected cells can be directly
resuspended in Flow Cytometry Buffer. If performing livecell analysis to maintain cells in culture after cell sorting, do
not use Fixation Buffer. Instead, resuspend cells directly in
Flow Cytometry Buffer and perform experiments.
24. It is also possible to monitor intracellular protein expression in
immune cells by flow cytometry. If this is desired, following
infection, detachment of immune cells, staining with a fixationcompatible viability dye, and surface antibody staining if
desired (through step 19 of Subheading 3.2), infected
immune cells are fixed using Fixation Buffer. Following fixation, fixed cells are permeabilized and then stained with fluorescent antibodies that recognize host proteins of interest.
After incubating with fluorescent antibodies, proceed from
step 20 as described in Subheading 3.2.
25. It is also possible to monitor additional features of the hostpathogen interaction using commercially available dyes
(ex. LysoSensor for pH, MitoSox for metabolic state)
[12, 13]. If this is desirable, the above protocol may need to
be modified in order to accommodate manufacturer-specific
protocols.
26. It is possible to analyze these samples using other techniques
such as mass cytometry or image cytometry. Mass cytometry
enables analysis of tens of proteins simultaneously on the same
cell; however, fluorescent mycobacteria cannot be identified
using this technique. Image cytometry, though lower throughput and fewer channels available for interrogation, enables the
analysis of protein localization in addition to expression
[4, 14].
27. FSC and SSC should be acquired on a linear scale to discriminate cells from debris. Alternative methods for doublet discrimination
are
available
including
acquiring
FSC-A vs. FSC-W, FSC-H vs. FSC-W, or SSC-A vs. SSC-W
among others.
Acknowledgments
This work was supported by the Ferry Fund (MIT), the Terry and
Susan Ragon Foundation, The Ragon Institute, and the MIT
Department of Biological Engineering.
270
Sydney L. Solomon and Bryan D. Bryson