Pegfp puro

Reagents utilized include: Silicone oil (Fisher S159-500), Borosilicate glass (Harvard Apparatus, type GC150T-10, 30-0062), Petrolatum (Sigma-Aldrich, 16415), Puromycin (BioShop, PUR333.100), Geneticin (Gibco, #11811023). Plasmids used for transfections: px459V2 (Addgene # 62988), pLVX-dTomato-C1 (Takara Bio), pEGFP-puro (Addgene #45561), or pBS/KSTau/LacZ (courtesy of Dr. Friedhelm Bladt, SLRI, Mt. Sinai Hospital, Toronto), modified from TauLacZ LTNL (Dr. Peter Mombaerts, Max Planck Institute, Frankfurt). Plasmids were prepared from bacterial stocks grown overnight using EndoFree Plasmid Maxi Kit (Qiagen, #12362), with plasmid integrity verified using gel electrophoresis and DNA transfection quality independently determined using electroporation and polyethylenimine (Sigma Aldrich, 25 kDa MW, # 408727) mediated transfection of HEK 293T cells (ATCC CRL-3216) according to standard methods together with culture lines and cell media constituents as indicated below. Analyses for beta-galactosidase activity in transfected cells were performed as previously described^{48 (link)}. Cell viability was assessed using standard trypan blue exclusion assay (Gibco 15250061)^{49 (link)}, DAPI (Sigma D9542), propidium iodide (Sigma P4170)^{50 (link)} or Calcein-AM/ethidium homodimer live/dead assay⁵¹ (Molecular Probes L-3224) methods as described previously.

The 4T1-mScarlet and 67NR-GFP cell lines were generated by transfection using a 1:4 ratio of plasmid DNA:FugeneHD reagent (Promega), according to the manufacturer’s instructions, followed by selection with 500 µg/mL geneticin (Fisher BioReagents) and 3 µg/mL puromycin (MP Biomedicals), respectively. pmScarlet-H-C1 was a gift from Dorus Gadella (Addgene plasmid # 85043). pEGFP-puro was a gift from Michael McVoy (Addgene plasmid # 45561). The MDA-MB-231-mScarlet cell line was generated by electroporation (Lonza) of the pmScarlet-C1 plasmid, according to the manufacturer’s instructions, and selection with 500 µg/ml geneticin. After two weeks of drug selection, cells were sorted (BD FACSAriaIIµ, BD Biosciences), collecting the subpopulations expressing high levels of mScarlet or GFP.