Fluorescence Microscopy of GFP-Labeled Cells

YAA3 cells were treated with 3.6 µM of 11β,13-dihydrolactucin or 12.5 µM of FK506 for 90 min, followed by incubation with or without 3 mM MnCl2 for further 90 min. On the last 10 min of incubation, 10 µg/mL of Hoechst 33,342 (Sigma) nuclear dye were added. Cells were washed and resuspended in 5 µL of 1,4-Diazabicyclo[2.2.2]octane (DABCO, triethylenediamine) DABCO solution (200 mM DABCO in 75% (v/v) glycerol, 25% (v/v) PBS) (Sigma-Aldrich). The preparations were monitored for GFP fluorescence as previously described [27 (link)] using a Zeiss Imager Z2 (Zeiss, Germany) fluorescence microscope. Photographs were taken with an Axiocam 506 mono (Zeiss). Three images were taken and analyzed for each sample, each one containing ≈ 600 individual cells. Images were analyzed using Fiji-ImageJ 1.53f (NIH, Bethesda, MD, USA).