3.5 Fluorescence
Microscopy
1. Transfer 10μL of resuspended cells to a microscope slide.
2. Gently spread into a thin layer using the edge of a cover slip and
allow to air-dry in the dark for 10 min.
3. Add 10μL of microscopy mounting medium optimized for
fluorescence imaging.
4. Place the cover slip over the sample, immobilize with adhesive
and allow to air-dry for 30 min.
5. Analyze with a fluorescence microscope using the appropriate
excitation and emission wavelengths for the fluorophore
being used.
3.6 Flow Cytometry
1. Transfer 190–195μL of PBS into a separate tube or 96-well
flat-bottom plate.
2. Add 5–10μL of the resuspended cells to the PBS. Gently
pipette up and down to mix the contents.
3. Analyze cell suspension with a flow cytometer using the appropriate excitation and emission wavelengths for the fluorophore
being used. Collect data for 10,000–50,000 cells at 500–1000
event/s.
4 Notes
1. In addition to 6-TreAz, three azido trehalose analogs
(2-TreAz, 3-TreAz, and 4-TreAz) were developed
[17]. Whereas 2-, 4-, and 6-TreAz incorporated into trehalose
glycolipids through an intracellular route in M. smegmatis
(SugABC-LpqY!Pks13/CmrA!MmpL3), 3-TreAz was
incorporated through an extracellular route (directly mycoloylated via Ag85) (see Fig. 1a). Together, these probes allow
pathway-targeted investigation of mycomembrane biosynthesis
and labeling of trehalose glycolipids.
2. At present, 6-TreAz and O-AlkTMM-C7 are commercially
available (Click Chemistry Tools). Other trehalose- and
TMM-based must be synthesized. The synthesis procedures
for FITC-Tre and DMN-Tre are described in Refs. 15 and
16. The synthesis procedures for other TMM-mimicking
probes are described in Refs. 22 and 24.
3. This procedure uses a final probe stock concentration of 5 mM,
although this can be adjusted as needed. Various azide-, alkyne-,
cyclooctyne-, and tetrazine-modified reagents (e.g., fluorophores, affinity tags) with different linkers are commercially
available. Follow manufacturer instructions for storage and
stock solution preparation. Picolyl azide- and picolyl alkynefunctionalized reagents [38] are also available, which exploit
copper chelation to raise the CuAAC reaction efficiency (up to
394
Nicholas Banahene and Benjamin M. Swarts
Microscopy
1. Transfer 10μL of resuspended cells to a microscope slide.
2. Gently spread into a thin layer using the edge of a cover slip and
allow to air-dry in the dark for 10 min.
3. Add 10μL of microscopy mounting medium optimized for
fluorescence imaging.
4. Place the cover slip over the sample, immobilize with adhesive
and allow to air-dry for 30 min.
5. Analyze with a fluorescence microscope using the appropriate
excitation and emission wavelengths for the fluorophore
being used.
3.6 Flow Cytometry
1. Transfer 190–195μL of PBS into a separate tube or 96-well
flat-bottom plate.
2. Add 5–10μL of the resuspended cells to the PBS. Gently
pipette up and down to mix the contents.
3. Analyze cell suspension with a flow cytometer using the appropriate excitation and emission wavelengths for the fluorophore
being used. Collect data for 10,000–50,000 cells at 500–1000
event/s.
4 Notes
1. In addition to 6-TreAz, three azido trehalose analogs
(2-TreAz, 3-TreAz, and 4-TreAz) were developed
[17]. Whereas 2-, 4-, and 6-TreAz incorporated into trehalose
glycolipids through an intracellular route in M. smegmatis
(SugABC-LpqY!Pks13/CmrA!MmpL3), 3-TreAz was
incorporated through an extracellular route (directly mycoloylated via Ag85) (see Fig. 1a). Together, these probes allow
pathway-targeted investigation of mycomembrane biosynthesis
and labeling of trehalose glycolipids.
2. At present, 6-TreAz and O-AlkTMM-C7 are commercially
available (Click Chemistry Tools). Other trehalose- and
TMM-based must be synthesized. The synthesis procedures
for FITC-Tre and DMN-Tre are described in Refs. 15 and
16. The synthesis procedures for other TMM-mimicking
probes are described in Refs. 22 and 24.
3. This procedure uses a final probe stock concentration of 5 mM,
although this can be adjusted as needed. Various azide-, alkyne-,
cyclooctyne-, and tetrazine-modified reagents (e.g., fluorophores, affinity tags) with different linkers are commercially
available. Follow manufacturer instructions for storage and
stock solution preparation. Picolyl azide- and picolyl alkynefunctionalized reagents [38] are also available, which exploit
copper chelation to raise the CuAAC reaction efficiency (up to
394
Nicholas Banahene and Benjamin M. Swarts