Plenti6.2 mtagbfp2

The open reading frames of mTagBFP2 and mNeongreen2 were amplified by PCR from donor vectors pBAD-mTagBFP2 (Addgene, #34632) and pSFFV_mNG2 (11)1–10 (Addgene, #82610) respectively, and ligated in a pLenti6.2 destination vector. Both vectors were verified by Sanger sequencing and deposited to Addgene as pLenti6.2_mTagBFP2 (#113725) and pLenti6.2_mNeonGreen2 (#113727).
Stable cell lines were generated using the lentiviral transduction of α cells and β cells using pLenti6.2_mTagBFP2 and pLenti6.2_mNeonGreen2, respectively. Both plasmids were co-transfected separately with third-generation lentiviral packaging and envelope vectors [pMD2. G (Addgene, #12259), pRSV-Rev (Addgene, #12253), and pMDLg/pRRE (Addgene, #12251)] into HEK-293T cells using the PEIpro (VWR) transfection reagent. After 24 h, the viral supernatant was collected and used to transduce α and β cells. Positive cells were selected 48 h after transduction using 1 μg/ml puromycin dihydrochloride (Sigma-Aldrich) added to growth media for at least 7 days. Transduction efficiency was assessed by fluorescence microscopy after a minimal culture period of 7 days.

Mouse alpha cells were labelled with BFP2 and rat beta cells were labelled with mNeonGreen to distinguish the different cell types. To generate the vectors, the open reading frames of mTagBFP2 and mNeongreen2 were amplified by PCR from donor vectors pBAD-mTagBFP2 (Addgene #34632) and pSFFV_mNG2(11)1-10 (Addgene #82610), respectively, and ligated in a pLenti6.2 destination vector. After verification by Sanger sequencing, they were deposited as pLenti6.2_mTagBFP2 (Addgene #113725) and pLenti6.2_mNeonGreen2 (Addgene #113727). The plasmids were then co-transfected separately with third-generation lentiviral packaging and envelope vectors: pMD2.G (Addgene #12259), pRSV-Rev (Addgene #12253), and pMDLg/pRRE (Addgene #12251) into HEK293ft cells (Thermo Fisher R70007) using the PEIpro (VWR) transfection reagent. After 24 h, the viral supernatant was collected in either alphaTC1 or INS1E medium and filtered. Stable cell lines were generated by diluting the lentivirus 1:10 and 1:20 in cell type–specific medium and transducing the alphaTC1 and INS1E using pLenti6.2_mTagBFP2 and pLenti6.2_mNeonGreen2, respectively. Selection with 1 μg/ml puromycin dihydrochloride (Sigma-Aldrich) began 48 h after transduction and continued for at least 7–14 days. The transduction efficiency was assessed by flow cytometry and was determined to be 66.9% for INS1E cells and 80.4% for alphaTC1 cells.