40-fold increase in signal intensity) and decrease toxicity (at least
10-fold reduction). Dibenzocyclooctyne (DBCO)-modified
reagents are commonly used for SPAAC reactions.
4. Various CuAAC ligands are commercially available, with TBTA
being the most commonly used at present. Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) is a water-soluble alternative to TBTA.
5. If the probe stock is in DMSO, then 198:2 dilution will result
in a final DMSO concentration of 1%. No-probe control
experiments should maintain an equal concentration
of DMSO.
6. The incubation times employed for efficient probe incorporation in M. smegmatis typically range from 0.5 to 16 h. However, the time of incubation depends on the objective of the
experiment and this parameter often needs to be optimized.
For example, shorter durations may be used to assess cellular
processes occurring on smaller time scales, while longer durations may be used to increase incorporation efficiency in slowgrowing species such as M. tuberculosis.
7. OD 600 can be measured over time to acquire a growth curve
and to determine whether the probe incubation step has an
impact on growth. At the probe concentrations reported to be
effective for metabolic labeling (typically 10–100μM), growth
inhibition has not been observed.
8. DMN-Tre is fluorogenic and thus its incorporation can be
analyzed by microscopy, and potentially other methods, without wash steps [16, 39].
9. Fluorescent probes are in vivo-compatible and analyses can be
performed on either live or fixed cells. However, it may be
desirable to fix the cells, for example to inactivate potentially
hazardous samples or to allow sample analysis at a later time.
For two-step “clickable” probes using CuAAC, due to copper
toxicity, it is usually preferable to fix cells with formaldehyde
prior to CuAAC, although in vivo-compatible CuAAC conditions using appropriate reagents and ligands have been developed [38, 40]. Copper-free SPAAC and tetrazine reactions are
inherently in vivo-compatible and analyses can be performed
on either live or fixed cells.
10. Different types of analyses can be performed on labeled cells or
cell extracts, depending on the experimental objective and the
nature of the metabolic labeling conditions. In most cases,
bacteria are fluorescently labeled via one-step or two-step
approaches, and cellular fluorescence analysis is done by
microscopy or flow cytometry, although other types of fluorescence analysis (e.g., microplate reader) are possible as well.
Procedures for performing microscopy and flow cytometry
Trehalose Probe Labeling
395
10-fold reduction). Dibenzocyclooctyne (DBCO)-modified
reagents are commonly used for SPAAC reactions.
4. Various CuAAC ligands are commercially available, with TBTA
being the most commonly used at present. Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) is a water-soluble alternative to TBTA.
5. If the probe stock is in DMSO, then 198:2 dilution will result
in a final DMSO concentration of 1%. No-probe control
experiments should maintain an equal concentration
of DMSO.
6. The incubation times employed for efficient probe incorporation in M. smegmatis typically range from 0.5 to 16 h. However, the time of incubation depends on the objective of the
experiment and this parameter often needs to be optimized.
For example, shorter durations may be used to assess cellular
processes occurring on smaller time scales, while longer durations may be used to increase incorporation efficiency in slowgrowing species such as M. tuberculosis.
7. OD 600 can be measured over time to acquire a growth curve
and to determine whether the probe incubation step has an
impact on growth. At the probe concentrations reported to be
effective for metabolic labeling (typically 10–100μM), growth
inhibition has not been observed.
8. DMN-Tre is fluorogenic and thus its incorporation can be
analyzed by microscopy, and potentially other methods, without wash steps [16, 39].
9. Fluorescent probes are in vivo-compatible and analyses can be
performed on either live or fixed cells. However, it may be
desirable to fix the cells, for example to inactivate potentially
hazardous samples or to allow sample analysis at a later time.
For two-step “clickable” probes using CuAAC, due to copper
toxicity, it is usually preferable to fix cells with formaldehyde
prior to CuAAC, although in vivo-compatible CuAAC conditions using appropriate reagents and ligands have been developed [38, 40]. Copper-free SPAAC and tetrazine reactions are
inherently in vivo-compatible and analyses can be performed
on either live or fixed cells.
10. Different types of analyses can be performed on labeled cells or
cell extracts, depending on the experimental objective and the
nature of the metabolic labeling conditions. In most cases,
bacteria are fluorescently labeled via one-step or two-step
approaches, and cellular fluorescence analysis is done by
microscopy or flow cytometry, although other types of fluorescence analysis (e.g., microplate reader) are possible as well.
Procedures for performing microscopy and flow cytometry
Trehalose Probe Labeling
395