Table 1.
Technical considerations for the use of IMPACT and RT-IMPACT for measurement of cellular PLD activities.
| Technical considerations | IMPACT | RT-IMPACT |
|---|---|---|
| Recommended types of applications (see Table 2 for details) | Determination of PLD activity at the single-cell or bulk population levels, amenable to either short- or long-term labeling timescales | Determination of organelle-level, subcellular localizations ofPLD activity, amenable to short-term labeling timescales |
| Detection methods (see Table 2 for details) | Flow cytometry or fluorescence microscopy (single-cell measurements); HPLC or mass spectrometry (bulk population measurements) | Fluorescence microscopy for real-time imaging; flow cytometry recommended to establish optimal labeling conditions depending on PLD stimulus |
| Reagents required for transphosphatidylation and click chemistry steps | (1) Recommended protocol: 3-azidopropanol and BCN-BODIPY (λex: 495; λem: 510) (2) Alternative protocol: 5-hexyn-1-ol and azido-fluorophores |
(S)-oxoTCO-C1 and Tz-BODIPY (λex: 490; λem 510) |
| Storage conditions for reagents | −20 °C (neat or in DMSO stocks; stable indefinitely) | −80°C (neat; stable indefinitely); —20°C (DMSO stock; use within a few months) |
| Stability of reagents in aqueous buffer/cell media | Stable indefinitely | Limited stability; use oxoTCO within 30 min and Tz-BODIPY within 4 h of dilution into aqueous buffer |
| Availability of reagents | (1) Recommended protocol: 3-azidopropanol is commercially available and moderate organic synthesis required to prepare BCN-BODIPY. (2) Alternative protocol: 5-hexyn-1-ol and azido-fluorophores are commercially available |
Complex, multistep organic synthetic sequences (involving photochemistry, flow chemistry, and pyrophoric reagents) are required to prepare oxoTCO and Tz-BODIPY |