The plasmids used for expression of all FPs in mammalian cells were based on pEGFP-N1 backbone (Clontech). To construct the plasmids (piRFP670-N1, piRFP682-N1, piRFP702-N1, piRFP713-N1, piRFP720-N1, pmNeptune-N1, pE2-Crimson-N1, peqFP650-N1, peqFP670-N1) the respective genes were PCR amplified as AgeI-NotI fragments and swapped with a gene of EGFP in pEGFP-N1.
To construct pMito-iRFP670, pMito-iRFP713, and pMito-iRFP720 iRFP670, iRFP713, and iRFP720 genes were cut from N1 plasmids and swapped with mTagBFP gene in pMito-mTagBFP22 (link). To generate pNLS-iRFP670, pNLS-iRFP713, and pNLS-iRFP720 iRFP670, iRFP713, and iRFP720 genes were cut from piRFPs-N1 plasmids AgeI-NotI fragments and swapped with a gene of ECFP in the pNLS-ECFP plasmid.
HeLa cell lines were grown in DMEM containing 10% FBS, 0.5% penicillin-streptomycin and 2 mM glutamine (Invitrogen). For microscopy cells were cultured in 35 mm glass bottom culture dishes with no. 1 cover glasses (MatTek). MTLn3 rat adenocarcinoma cells were cultured in αMEM medium (Life Technologies) supplied with 5% FBS, 0.5% penicillin-streptomycin, and 2 mM glutamine (Life Technologies). Plasmid transfections were performed using an Effectene reagent (Qiagen) according to the manufacturer’s protocol. Stably-expressing cells were selected with 700 mg/ml G418 antibiotic. Sorting of positive cells was performed using a MoFlo XDP sorter (Beckman Coulter) equipped with a 676 nm Kr laser and a 700 nm LP emission filter.
To construct pMito-iRFP670, pMito-iRFP713, and pMito-iRFP720 iRFP670, iRFP713, and iRFP720 genes were cut from N1 plasmids and swapped with mTagBFP gene in pMito-mTagBFP22 (link). To generate pNLS-iRFP670, pNLS-iRFP713, and pNLS-iRFP720 iRFP670, iRFP713, and iRFP720 genes were cut from piRFPs-N1 plasmids AgeI-NotI fragments and swapped with a gene of ECFP in the pNLS-ECFP plasmid.
HeLa cell lines were grown in DMEM containing 10% FBS, 0.5% penicillin-streptomycin and 2 mM glutamine (Invitrogen). For microscopy cells were cultured in 35 mm glass bottom culture dishes with no. 1 cover glasses (MatTek). MTLn3 rat adenocarcinoma cells were cultured in αMEM medium (Life Technologies) supplied with 5% FBS, 0.5% penicillin-streptomycin, and 2 mM glutamine (Life Technologies). Plasmid transfections were performed using an Effectene reagent (Qiagen) according to the manufacturer’s protocol. Stably-expressing cells were selected with 700 mg/ml G418 antibiotic. Sorting of positive cells was performed using a MoFlo XDP sorter (Beckman Coulter) equipped with a 676 nm Kr laser and a 700 nm LP emission filter.
