A7888

The channel was first passivated with a 7.5 mg/mL bovine serum albumin (Sigma-Aldrich, A7888) solution for 2 h to further inhibit Taq adsorption, and flushed with DI water at 1 μL/min for 30 min to remove unbound particles. During the test, a 20 μL paraffin oil plug (VWR, BDH3338) was pumped through the chip, followed by a 10 μL sample, and then another oil plug. The two plugs prevent sample evaporation caused by heating32 (link).
Unless specified, samples contained an initial DNA concentration of 10⁵ copies/μL and tests were conducted with a syringe pump (New Era, NE-1000) set at 1 μL/min. Due to absorption of the oil into the PDMS, the actual flow rate was calculated to be 0.8 μL/min based on the time the samples took to go from the inlet to the outlet.

Pancreatic islets were obtained from 8-week-old male C57B6 mice, using collagenase (Librase; Roche, Switzerland) digestion [18 (link)]. After digestion, healthy round islets were hand-picked under a stereoscopic microscope, then incubated for 12 hours in RPMI1640 medium (Nacalai Tesque, Japan) containing 7 mM glucose supplemented with 10% v/v fetal bovine serum (FBS; Invitrogen/Thermo Fisher Scientific, USA). Twenty-four hours before the secretion experiments, the islets were divided into 2 groups and placed in RPMI media containing different concentration of glucose (7 or 15 mM) and supplemented with 10% v/v FBS. For the assessment of glucagon and insulin secretion, batches of 20 healthy size-matched islets were preincubated for 30 min in HEPES-balanced Krebs-Ringer bicarbonate (KRB) buffer containing 0.1% bovine serum albumin (BSA; A-7888, Sigma-Aldrich, U.S.A.) and 1 mM glucose. Subsequently, the islets were incubated for 60 min in KRB buffer containing 0.1% BSA with different glucose concentrations (1, 7, or 25 mM). The supernatants of the incubation buffers were used for the insulin and glucagon assays. Glucagon concentrations were measured using specific ELISA for glucagon (Mercodia, Sweden) immediately after the experiments, and insulin concentrations were measured by ELISA for insulin (Morinaga, Japan).

Duplicate samples and standards in coating buffer (0,012M NaCO₃ and 0,028M NaHCO₃, pH 9,6) were incubated overnight at 4°C. Samples were diluted 1:100 after titration was performed. Custom made autotaxin (ENPP2-8H, AGF06181012)(Ascent Gene, MD, USA) at concentrations (100–3.125) ng/ml was used to construct a linear standard curve. After blocking for 1.5 hours with 1.5% BSA in PBS-T, samples were incubated with a-ATX rabbit anti-mouse antibody 1:1000 (10005373, Cayman, Tallinn, Estonia) for 1 hour. The a-ATX antibody was detected with an a-rabbit HRP conjugated antibody 1:2000 (4010–05, Southern Biotech, AL, USA). Colour was developed using TMB (3,3’,5,5’-Tetramethylbenzidine, A7888, Sigma, USA). The reaction was stopped with 2M H₂SO₄ and readings were obtained at 450nm.