Novolog

Apidra (Sanofi, Bridgewater, NJ ), Humalog (Eli Lilly & Co., Indianapolis, IN), and Novolog (Novo Nordisk, Plainsboro Township, NJ) purchased from a pharmacy were passed through spin Zeba desalting columns (Thermo Fisher Scientific, Waltham, MA) to remove phenolic compounds [22 (link)]. The column was placed in a 1.5–2.0mL collection tube and centrifuged at 1500 × g for 1 minute to remove storage solution. Next, 300μL of PBS was passed through the column by centrifugation at 1500 × g for 1 minute. After washing the resin 3 times, the column was placed in a new collection tube and commercial insulin was applied to the top of the compact resin bed. Insulin was eluted by centrifugation at 1500 × g for 2 minutes and collected. A standard protein assay (Thermo Fisher Scientific, Waltham, MA) was performed on the post-column eluates to confirm retrieval of insulin protein following removal of phenolic compounds.

MTT assay is widely used to show loss of cellular metabolic activity (ATCC bioproducts, Manassas, VA). Cells undergoing analysis via the cytotoxicity assay were plated in a 48-well plate with 2.5x10⁵ cells/well. These cells were incubated overnight in 250µl of the growth media required for each individual cell line. Mouse cell lines and PBMCs were exposed to saline, sterile diluent (Eli Lilly and Company, Indianapolis, IN) and Humalog (Eli Lilly and Company, Indianapolis, IN) at a serial dilution of 1:3 for 1 and 3-days. PMBCs were also exposed to Apidra (Sanofi, Bridgewater, NJ ), Humalog (Eli Lilly & Co., Indianapolis, IN), and Novolog (Novo Nordisk, Plainsboro Township, NJ) with and without phenolic compounds at a serial diluent of 1:3 for 1-day. Following an incubation period of 3 days for PBMC and 1- and 3-days for mouse cell lines, 30µl of MTT reagent was added to each well and plates were incubated for 1 hour at 37°C until visibility of purple precipitate. Next, all supernatants were discarded and 300µl DMSO was added, mixed well, and 100µl samples were transferred to a 96-well plate in duplicates. Absorbance was read at 570 nm for analysis.