Multiscan spectrum spectrophotometer

Corn starch, potato starch, soluble starch, dextran, pullulan, bacterial α-1,3-glucan, nigeran, cellulose, pustulan, and laminaran were tested as rAoAgtA substrates. A mixture (12 μL) containing 2 mg/mL (0.2%) each substrate and 24 mU/mL purified rAoAgtA in 50 mM Na-Ac buffer (pH 5.5) was incubated at 40°C for 4 h. Then, H₂O (138 μL) was added, and the reaction was immediately stopped by boiling for 5 min. The amount of reducing sugar generated from each substrate was measured by bicinchoninic acid method (Doner and Irwin, 1992 (link); Utsumi et al., 2009 (link)). The bicinchoninic acid working reagent was prepared according to Utsumi et al. (2009) (link), and 100 μL was added to a 100-μL aliquot of each boiled reaction mixture. The sample was incubated at 80°C for 40 min and then at room temperature for 15 min, and the absorbance at 560 nm was measured with a Multiscan Spectrum spectrophotometer (Thermo Fisher Scientific). One unit of enzyme activity was defined as the amount of enzyme required to liberate 1 μmol of reducing sugars (D-glucose conversion) from the substrate per minute. The detection limit was 1 mU/mL.

Cell viability was assessed using Alamar blue reagent (Invitrogen, CA, USA). MCAS and OVSAHO cells were seeded in 96 well culture plates at a density of 10⁴ cells per well, and transfected 1 day later with FAT4 siRNA, and incubated for 24–96 h at 37 °C and 5% CO₂. Cell proliferation was measured by adding 10 μl Alamar blue dye to the culture medium and incubating for 4 h at 37 °C. The absorbance was measured at 570 nm using Multiscan spectrum spectrophotometer (Thermo Fisher Scientific Inc., MA, USA).

Following defrosting of the hemolymph samples, 2 μL of hemolymph/anticoagulant buffer solution was added to 500 μL of PBS (pH 7.4). 100 μL of this solution was placed in a well of a 96‐well microplate with 100 μL of 10 mmol/L dopamine. While many researchers use L‐dopa as a substrate for PO reactions, for insect POs, dopamine is the preferred substrate over L‐dopa. It is the natural substrate for insects and is more soluble than L‐dopa (Sugumaran 1998). Readings were taken every 10 sec for 3 min at 490 nm and 25°C on a Thermo Scientific Multiscan Spectrum spectrophotometer. The maximum rate of reaction across six windows of change (absorbance readings) was then used as an approximation of PO level.