The setup of the FLIM system consisted of a picosecond diode laser (laser power, 5 mW) with an emission wavelength of 470 nm (LDH470; PicoQuant, Berlin, Germany) and a ~70 ps pulse width for the excitation of o-BMVC under a scanning microscope (IX-71 and FV-300; Olympus, Tokyo, Japan). The fluorescent signal from o-BMVC was collected using a 60× NA = 1.42 oil-immersion objective (PlanApoN; Olympus, Japan), passing through a 550/88 nm bandpass filter (Semrock, Rochester, NY, USA), followed by detection using a SPAD (PD-100-CTC; Micro Photon Devices, Bolzano, Italy). The fluorescence lifetime was recorded and analyzed using a time-correlated single-photon counting (TCSPC) module and software (PicoHarp 300 and SymPhoTime v5.3.2; PicoQuant, Berlin, Germany). FLIM images were constructed from pixel-by-pixel lifetime information.
For the study of G4 ligands pretreatment, 10 µM TMPyP4, BMVC4 and BRACO-19 were used for pretreatment of HeLa cells and H33258 for pretreatment of MRC-5 cells. After washing twice, cells were fixed with 70% ethanol for 10 min and then stained with 5 µM o-BMVC for 10 min at room temperature. Quantitative analysis of o-BMVC foci by using the Otsu algorithm for the image analysis was described previously [10 (link)].
For the study of G4 ligands pretreatment, 10 µM TMPyP4, BMVC4 and BRACO-19 were used for pretreatment of HeLa cells and H33258 for pretreatment of MRC-5 cells. After washing twice, cells were fixed with 70% ethanol for 10 min and then stained with 5 µM o-BMVC for 10 min at room temperature. Quantitative analysis of o-BMVC foci by using the Otsu algorithm for the image analysis was described previously [10 (link)].
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