Infinite f500

The rest of the transfected cells used for ß-arrestin assay (Fig. 5A,C,E) were plated to a 96 well plate after 24 hours from the transfection. 48 hours later from the transfection, the cells were fixed by 50/50% methanol/acetone for 1 min at room temperature and permeabilized by 0.2% TritonX-100 for 9 minutes after several washes by PBS, the cells were blocked with 3% BSA for 1 hour at room temperature. Then cells were incubated with indicated primary antibody (1:1000 dilution anti-HA; 16B12 mouse 901514 Biolegend, 1:1000 dilution anti-Flag; F3165 mouse SIGMA, 1:10000 dilution anti-GFP; rabbit ab290 abcam) in 3% BSA for overnight at 4 °C. After washing the plate 4 times with PBS, plate was incubated with 50 µL/well of secondary antibody (1:20000 dilution; HRP conjugated anti-mouse IgG DC02L Millipore, 1:20000 dilution; HRP conjugated anti-rabbit antibody A0545 Sigma) for 1 hour at room temperature. After washing the plate 6 times with PBS, 50 µL/well of LuminataTM Crescendo ELISA HRP Substrate (Millipore) were added and the luminescence were read by TECAN Infinite F500 (Tecan Group, Ltd., Männedorf, Switzerland).

Ten thousand cells were seeded in 100 μl of media in each well of a 96-well flat-bottom transparent plate. One-tenth of the volume of AlamarBlue reagent (G-Biosciences, 786–921) was directly added to the wells and incubated for 4 hr at 37°C in a cell culture incubator, shielded from direct light. Results were recorded by measuring fluorescence using a fluorescence excitation wavelength with a peak excitation of 570 nm and a peak emission of 585 nm on a microplate reader (Tecan Infinite F500, Tecan Group Ltd.).

The total starch content of feed and digesta samples was determined according to the AOAC Method 996.11 (KOH format) [36 (link)] using the Total Starch Assay Kit (Megazyme, Bray, Ireland) as modified by Martens et al. (2018) [37 (link)]. In brief, 25 μL supernatant of enzymatically treated samples was transferred in the wells of a 96 well plate followed by the addition of 225 μL glucose oxidase peroxidase (GOPOD) reagent (Megazyme). The reaction was performed in a shaking incubator at 50 °C for 20 min, and the absorbance at 520 nm was read against reagent blank using a Tecan Infinite^® F500 (Tecan Group Ltd., Männedorf, Switzerland) spectrophotometer. The glucose (Glc) content was determined using a Glc calibration curve (0.1–0.6 mg/mL).

Intracellular ROS were evaluated with DCFDA (2′,7′-dichlorofuorescein diacetate) (Sigma Aldrich, D6883, St. Louis, MO, USA), a membrane permeable probe that becomes fluorescent when tied to ROS [34 (link)]. βtc3 cells were pre-loaded with 15 μM DCFDA in Krebs–Ringer buffer (125 mM NaCl, 5 mM KCl, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 25 mM HEPES-NaOH pH 7.4 and 2 mM CaCl₂) supplemented with 11 mM glucose for 1 h at 37 °C. The ROS content was detected for 30 min both in basal and stress conditions with a microplate reader (485/528 nm Ex/Em) (TECAN Infinite^® F500, Tecan Group Ltd. Männedorf, Switzerland). Mean values and standard deviations were based on three independent experiments.

IP formation was quantified with HTRF (Homogeneous Time-Resolved Fluorescence) based “Cisbio IP-One Tb” (Cisbio, Bagnols-sur-Cèze, France) assay kit, following manufacturer’s instructions. EndoC-βH2 cell suspensions (5 × 10⁴ cells) were treated in 384-well plate (16 μl volume) with modified stimulation buffer (10 mM Hepes, 10 mM MES, 1 mM CaCl2, 0.5 mM MgCl₂, 4.2 mM KCl, 146 mM NaCl, 5.5 mM glucose, 50 mM LiCl) at pH 7.4 or 6.4 for 60 min at 37 °C. Where indicated, cells were pretreated with YM-254890, a selective Gα_q/11 inhibitor27 for 30 min prior to incubation with the IP stimulation buffer and maintained throughout the IP determination. IP measurements were performed in triplicates and experiments were repeated at least three times. Samples were read on a TECAN Infinite F500 (Tecan Group, Ltd., Männedorf, Switzerland) with excitation at 320 nm and emission at both 620 nm and 665 nm.
cAMP activity was measured using a cAMP-HTRF assay kit (Cisbio) following manufacturer’s instructions. EndoC-βH2 cells (5 × 10³) were treated in 384-well plate (12 μl final volume) with stimulation buffer (PBS containing 10 mM of each HEPES, MES, and 0.5 mM IBMX, at pH 7.4 or 6.4) for 30 min at room temperature. Cells were lysed using kit lysis buffer and cAMP was then measured in 384 well plates (HTRF) with TECAN Infinite F500.

For BRET donor saturation curves were obtained as previously described^{59 (link)}, HEK293 cells seeded in 24-well plates were transiently transfected with 3 ng of GPR61-Rluc, 0.8 ng of GPR62-Rluc, 0.2–0.4 ng of GPR135-Rluc and 5–400 ng of the corresponding YFP plasmids. 24 h after transfection, cells were transferred into a 96-well white Optiplate (Perkin Elmer Life Sciences) precoated with 10 μg/ml poly-L-lysine (Sigma) and incubated for another 24 h before BRET measurements. Luminescence and fluorescence were measured simultaneously using the TECAN Infinite F500 (Tecan Group, Ltd., Männedorf, Switzerland).