1. Follow steps 1–7 in Subheading 3.1.
2. Add 2μL of 5 mM FITC-Tre, DMN-Tre, or O-FITC-TMM
(stocks prepared as described in Subheadings 2.1 and 2.2), or
other fluorophore-conjugated trehalose analog (or vehicle control) to 198μL bacteria. Mix the well contents.
3. Continue through steps 8–13 in Subheading 3.1 (see Note 8).
4. Optionally fix the cells (see Note 9):
(a) Resuspend cells in 150μL of 4% formaldehyde in PBS.
(b) Cover the plate and incubate in the dark for 10 min.
(c) Pellet the cells by centrifugation and wash three times as
described in steps 10–13 of Subheading 3.1.
5. Resuspend the cells in 200μL PBS.
6. Prepare cells for fluorescence microscopy or flow cytometry
analysis as described in Subheadings 3.5 or 3.6, respectively
(see Note 10).
3.3 Two-Step
Labeling: CuAAC
“Click” Reaction
This procedure is applicable to metabolic labeling experiments with
6-TreAz (or isomeric TreAz analogs), N-AlkTMM-C7,
O-AlkTMM-C7, O-AzTMM-C10, and other CuAAC-capable trehalose analogs. This procedure uses a final probe concentration of
50μM, although this can be adjusted as needed.
1. Follow steps 1–7 in Subheading 3.1.
2. Add 2μL of 5 mM 6-TreAz, O-AlkTMM-C7, N-AlkTMM-C7,
or O-AzTMM-C10 (stocks prepared as described in Subheadings 2.1 and 2.2), or other CuAAC-capable trehalose analog
(or vehicle control) to 198μL bacteria. Mix the well contents.
3. Continue through steps 8–13 in Subheading 3.1.
4. Fix and wash the cells as described in step 4 of Subheading 3.2
(see Note 9).
5. Resuspend the cells in 138μL PBS.
6. Sequentially add 3μL each of the following (stocks for each
prepared as described in Subheading 2.4):
(a) 1 mM Az-488or Alk-488.
(b) 60 mM freshly prepared sodium ascorbate.
(c) 6.4 mM TBTA.
(d) 50 mM copper sulfate.
7. Gently pipette up and down to mix reagents.
8. Cover the plate with aluminum foil.
9. Incubate in the dark for 30 min at room temperature.
10. Pellet cells by centrifugation and wash three times as described
in steps 10–13 of Subheading 3.1 to remove excess reagents.
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Nicholas Banahene and Benjamin M. Swarts
2. Add 2μL of 5 mM FITC-Tre, DMN-Tre, or O-FITC-TMM
(stocks prepared as described in Subheadings 2.1 and 2.2), or
other fluorophore-conjugated trehalose analog (or vehicle control) to 198μL bacteria. Mix the well contents.
3. Continue through steps 8–13 in Subheading 3.1 (see Note 8).
4. Optionally fix the cells (see Note 9):
(a) Resuspend cells in 150μL of 4% formaldehyde in PBS.
(b) Cover the plate and incubate in the dark for 10 min.
(c) Pellet the cells by centrifugation and wash three times as
described in steps 10–13 of Subheading 3.1.
5. Resuspend the cells in 200μL PBS.
6. Prepare cells for fluorescence microscopy or flow cytometry
analysis as described in Subheadings 3.5 or 3.6, respectively
(see Note 10).
3.3 Two-Step
Labeling: CuAAC
“Click” Reaction
This procedure is applicable to metabolic labeling experiments with
6-TreAz (or isomeric TreAz analogs), N-AlkTMM-C7,
O-AlkTMM-C7, O-AzTMM-C10, and other CuAAC-capable trehalose analogs. This procedure uses a final probe concentration of
50μM, although this can be adjusted as needed.
1. Follow steps 1–7 in Subheading 3.1.
2. Add 2μL of 5 mM 6-TreAz, O-AlkTMM-C7, N-AlkTMM-C7,
or O-AzTMM-C10 (stocks prepared as described in Subheadings 2.1 and 2.2), or other CuAAC-capable trehalose analog
(or vehicle control) to 198μL bacteria. Mix the well contents.
3. Continue through steps 8–13 in Subheading 3.1.
4. Fix and wash the cells as described in step 4 of Subheading 3.2
(see Note 9).
5. Resuspend the cells in 138μL PBS.
6. Sequentially add 3μL each of the following (stocks for each
prepared as described in Subheading 2.4):
(a) 1 mM Az-488or Alk-488.
(b) 60 mM freshly prepared sodium ascorbate.
(c) 6.4 mM TBTA.
(d) 50 mM copper sulfate.
7. Gently pipette up and down to mix reagents.
8. Cover the plate with aluminum foil.
9. Incubate in the dark for 30 min at room temperature.
10. Pellet cells by centrifugation and wash three times as described
in steps 10–13 of Subheading 3.1 to remove excess reagents.
392
Nicholas Banahene and Benjamin M. Swarts