A highly stable monomeric red fluorescent protein for advanced microscopy

To quantify the monomeric state of the selected FPs, we followed the OSER assay protocol described previously​⁹​. Briefly, HeLa cells were cultured in the same condition as described above. Cells were seeded on glass-bottom 24-well plates pre-coated with Matrigel (356235, BD Biosciences) 3 hours before transfection. 500 ng of each pCytERM-FPs plasmid were transfected at 40-50% confluency using Lipofectamine 3000 (Invitrogen, USA) or Hieff Trans Liposomal Transfection Reagent (Yeasen, 40802ES02) according to the manufacturer’s protocol. The experimenter was blinded to the plasmid identities, as all plasmid vials were barcoded, and their identities were revealed only after data collection and analysis were complete. Samples were transfected and imaged in a random order, with plasmid vials pooled together and randomly selected during transfection. Each independent transfection was also randomized separately. Transfected live cells were imaged on a CSU-W1 SoRa imaging setup of Nikon Spinning-Disk Field Scanning Confocal System (Nikon, Japan) 20-24 hours after transfection.  Image analysis was performed by three blinded independent researchers, using NIS Elements software, and mean values with standard errors were calculated. Positive cells selected for analysis include cells with clear nuclear structure, dead or highly stressed, and out-of-focus cells were excluded from analysis. Cells with non-spherical nuclei or condensed nuclei were counted as stressed cells and excluded from normal cells fraction.