Nanodrop system

INS-1E cells or human islets were treated with dantrolene and sitagliptin for 24 hours. In addition dantrolene and sitagliptin were kept in KRB buffer during GSIS. INS-1E cells were incubated in KRB buffer with 0 mM glucose for 40 minutes at 37 degrees. Cells were then incubated with KRB buffer and 2.8 mM glucose for 1 hour followed by KRB with 16.7 mM glucose for 1 hour at 37 degrees. Supernatant was collected for insulin quantification at the end of each incubation. Insulin levels were quantified using the Rat/Mouse insulin ELISA kit from EMD Millipore. Cells were lysed at the end of the experiment and protein was quantified using a NanoDrop system from Thermo Scientific. Data was presented as total insulin level normalized to total protein.
Human islets from 3 different donors were hand picked and 8–10 islets were transferred to an eppendorf tube. Islets were incubated in KRB buffer with 3.3 mM glucose for 40 minutes. Islets were then exposed to either 3.3 mM glucose or 16.7 mM glucose KRB buffer and after one hour buffer was collected for insulin analysis. Insulin levels were measured using the Stellux human insulin ELISA kit from ALPCO. DNA was extracted from islets using the Qiagen DNeasy Blood and Tissue Kit per protocol. DNA was quantified using a NanoDrop system from Thermo Scientific. Data was presented as total insulin level normalized to total DNA.

Primary human tracheobronchial basal cells were isolated and expanded in epithelial cell culture medium containing Y-27632 for two passages before transduction with either GFP- or mCherry-containing viruses (MOI = 100). Transduction was performed in culture medium plus 4 μg/mL polybrene and medium was exchanged for fresh culture medium after 16 h. Purification of cultures to remove untransduced cells was performed by FACS for GFP or mCherry positivity after 7–10 days of further expansion. Since both the GFP and mCherry lentiviral plasmids carry a puromycin resistance cassette, PCR copy number analysis for the puromycin cassette was performed to quantify the number of lentiviral copies incorporated per donor cell (Taqman custom copy number assay). Genomic DNA (gDNA) was extracted from each transduced cell culture using a blood & cell culture DNA mini kit (Qiagen) as per the manufacturer’s instructions. gDNA was quantified using a NanoDrop system and PCR performed using the TaqMan genotyping master mix and reference assay (Life Technologies). Stable transduction was confirmed by flow cytometry prior to competitive proliferation assays.

Each resected specimen was sectioned into five slices (8‐μm‐thick slices), and macroscopic trimming was performed to obtain as many cancer cells as possible for more than 50% tumor cellularity. Genomic DNA was extracted from formalin‐fixed paraffin‐embedded (FFPE) tissue samples using a GeneRead DNA FFPE Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Genomic DNA was extracted from the blood samples using a genomic DNA extraction kit (Katayama Chemical, Osaka, Japan). The concentration and purity of genomic DNA samples were determined using a NanoDrop system (Life Technologies, Carlsbad, CA, USA) and Qubit dsDNA HS Assay Kit (Life Technologies) designed to be accurate for sample concentrations of 10–100 ng/mL. Genomic DNAs from the FFPE tissue and blood samples were stored at −80°C until analysis.