7. Alternative buffers for cell detachment are acceptable for this
step (Trypsin, Accutase, etc.). Selection of detachment buffers
should be driven by experimental needs. In the event of analysis
of immune cell surface epitopes, it is preferred to minimize use
of enzymatic buffers.
8. Alternative technologies for cell counting such as automated
cell counters can be used.
9. Cells can be prepared directly in round-bottom tubes, if
desired.
10. Care should be taken when selecting viability dyes for immune
cells. Attention should be placed on whether the cells will be
analyzed in a fixed or unfixed environment as certain viability
dyes for immune cells are not compatible with Fixation Buffer.
11. It is possible to analyze mycobacterial samples by flow directly
from medium at this stage provided that there is access to a
flow cytometer in the BSL3 or a cytometer outside of the BSL3
that can accommodate live bacterial samples. In the event that
this is a possibility, resuspend mycobacterial cultures by pipetting and transfer appropriate volume directly to round-bottom
tubes.
12. 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, mycobacteria can be directly
resuspended in Flow Cytometry Buffer.
13. Currently, there is no consensus on how to perform viability
staining of live vs dead mycobacteria by flow cytometry. New
viability dyes have become available to attempt to perform this
measurement [8, 9]. To date, these dyes have not been applied
to mycobacteria in conjunction with cell sorting and plating for
viability or examination by electron microscopy in order to
obtain orthogonal validation of these dyes.
14. It is possible to detect proteins expressed on the mycobacterial
cell wall using a surface staining protocol that makes use of
fluorescent antibodies that recognize your epitope of interest
[10, 11]. If this is desired, following centrifugation of mycobacteria, remove the supernatant (after step 2 of Subheading
3.1, and resuspend mycobacteria in Flow Cytometry Buffer
containing fluorescent antibodies that recognize your epitope
of interest. After incubation with fluorescent antibodies, mycobacteria are washed at least twice with at least 1 mL of Flow
Cytometry Buffer prior to proceeding with step 3 of Subheading 3.1.
15. Given the difficulties in determining mycobacterial doublets by
flow cytometry, at this point, it is possible to filter samples
through a 5-μm filter to create a single-cell suspension; however, filtration can result in significant cell loss.
268
Sydney L. Solomon and Bryan D. Bryson
step (Trypsin, Accutase, etc.). Selection of detachment buffers
should be driven by experimental needs. In the event of analysis
of immune cell surface epitopes, it is preferred to minimize use
of enzymatic buffers.
8. Alternative technologies for cell counting such as automated
cell counters can be used.
9. Cells can be prepared directly in round-bottom tubes, if
desired.
10. Care should be taken when selecting viability dyes for immune
cells. Attention should be placed on whether the cells will be
analyzed in a fixed or unfixed environment as certain viability
dyes for immune cells are not compatible with Fixation Buffer.
11. It is possible to analyze mycobacterial samples by flow directly
from medium at this stage provided that there is access to a
flow cytometer in the BSL3 or a cytometer outside of the BSL3
that can accommodate live bacterial samples. In the event that
this is a possibility, resuspend mycobacterial cultures by pipetting and transfer appropriate volume directly to round-bottom
tubes.
12. 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, mycobacteria can be directly
resuspended in Flow Cytometry Buffer.
13. Currently, there is no consensus on how to perform viability
staining of live vs dead mycobacteria by flow cytometry. New
viability dyes have become available to attempt to perform this
measurement [8, 9]. To date, these dyes have not been applied
to mycobacteria in conjunction with cell sorting and plating for
viability or examination by electron microscopy in order to
obtain orthogonal validation of these dyes.
14. It is possible to detect proteins expressed on the mycobacterial
cell wall using a surface staining protocol that makes use of
fluorescent antibodies that recognize your epitope of interest
[10, 11]. If this is desired, following centrifugation of mycobacteria, remove the supernatant (after step 2 of Subheading
3.1, and resuspend mycobacteria in Flow Cytometry Buffer
containing fluorescent antibodies that recognize your epitope
of interest. After incubation with fluorescent antibodies, mycobacteria are washed at least twice with at least 1 mL of Flow
Cytometry Buffer prior to proceeding with step 3 of Subheading 3.1.
15. Given the difficulties in determining mycobacterial doublets by
flow cytometry, at this point, it is possible to filter samples
through a 5-μm filter to create a single-cell suspension; however, filtration can result in significant cell loss.
268
Sydney L. Solomon and Bryan D. Bryson