Fluorescent technologies enable the measurement of protease activities and the screening of compounds influencing protease activities. Fluorescence Resonance Energy Transfer (FRET) but also TR-FRET, Q-FRET together with Time Resolved Fluorescence (TRF) and Fluorescence Lifetime (FLT) are popular assays, in which protease substrates occupy a central place. These assays are based on the specific recognition and cleavage of a peptide sequence (substrate) by the protease of interest. If the substrate is not optimally designed (or too short), experimental output can be unrelevant: low selectivity and specificity, high background, false negatives or positives…). In addition, the data can also be affected by the reaction buffer (pH, compound solvent…).This lack of relevant biological information is at the origin the emergence of “Next-Gen” fluorescent protease substrates.
Intracellular Flow Cytometry enables the identification and analysis of signaling and functional markers within cells from samples containing heterogeneous cell populations. Many cell types can be thus identified through their intracellular Flow Cytometry patterns and key intracellular signaling pathways and their respective post-translational modifications can be conveniently analysed. Behind these unique performances, Intracellular Flow Cytometry require anyhow optimized immunoreagents for cell permeabilization, intracellular staining and fluorescent detection.