Human proinsulin

Human Insulin (Mercodia; Cat # 10-1113-01), human proinsulin (Mercodia; Cat # 10-1118-01), mouse insulin (Crystal Chem; Cat # 90080), mouse proinsulin (Mercodia; Cat # 10-1232-01), mouse glucagon (Crystal Chem; Cat # 81518), mouse LEGENDplex custom 10-plex (Biolegend) were measured using ELISA kits following manufacturer’s instructions.

Spent media collected from several time points were centrifuged to remove cell debris and frozen at −80 °C until being assayed. Before freezing, the pH was measured (Fisher Scientific, Cat# 13–636-AB15). Glucose and lactate were measured using a Stat Profile pHOx Ultra blood gas analyzer (Nova Biomedical). Nutrient consumption or metabolite production rates were calculated using the following equation: $$Q_{\text{glu}}\text{or}{Q}_{\text{lac}}=S_f-S_0/\left(\left[N/V_f\right]\times ▵t\right)$$ where Q_glu or Q_lac refers to glucose consumption or lactate production rate, respectively. S_f and S₀ are the concentrations of glucose or lactate in the spent and fresh media, respectively. N and V_f refer to the cell number and the media volume at the time of collection, respectively. Δt is the time between media change.
Amino acid concentrations were measured using high-performance liquid chromatography. Hormone levels were measured using the following ELISA kits: human proinsulin (Mercodia, Cat# 10-1118-01) and human C-peptide (ALPCO, Cat #80-CPTHU-CH01).