For characterization in cell suspension, 2–10 µm staurosporine (STS) was added to cells grown in 6‐well plates for 6 h before the analysis. Fluorescence spectra of the untreated (uncleaved) and staurosporine‐treated (cleaved) cell suspensions were recorded using 610 nm excitation and normalized by acceptor fluorescence at 670 nm for comparison.
For fluorescence microscopy, cells were cultured in 35‐mm glass‐bottom Petri dishes with no. 1 coverglass (MatTek). Live HeLa cells were imaged on Olympus IX81 inverted epifluorescence microscope operated with SlideBook v.4.1 software (Intelligent Imaging Innovations) and equipped with a 60 × 1.35 numerical aperture (NA) oil objective lens (UPlanSApo, Olympus) and an opiMOS sCMOS camera (QImaging). During imaging, HeLa cells were incubated in a cell imaging medium (Life Technologies‐Invitrogen) and kept at 37 °C. For FRET, a 605/30 excitation filter and two emission filters (667/30 nm for miRFP670 and 725/40 nm for iRFP720) were used. Fluorescence intensity ratio between FRET and donor was calculated. FRET measurements were quantified using ImageJ (NIH). Time‐course ratio measurements were normalized to baseline prestimulation values.
For fluorescence microscopy, cells were cultured in 35‐mm glass‐bottom Petri dishes with no. 1 coverglass (MatTek). Live HeLa cells were imaged on Olympus IX81 inverted epifluorescence microscope operated with SlideBook v.4.1 software (Intelligent Imaging Innovations) and equipped with a 60 × 1.35 numerical aperture (NA) oil objective lens (UPlanSApo, Olympus) and an opiMOS sCMOS camera (QImaging). During imaging, HeLa cells were incubated in a cell imaging medium (Life Technologies‐Invitrogen) and kept at 37 °C. For FRET, a 605/30 excitation filter and two emission filters (667/30 nm for miRFP670 and 725/40 nm for iRFP720) were used. Fluorescence intensity ratio between FRET and donor was calculated. FRET measurements were quantified using ImageJ (NIH). Time‐course ratio measurements were normalized to baseline prestimulation values.