Ip one elisa kit

Frozen tissue was weighed and sonicated in 10 μL/mg of tissue of Tris-HCl buffer (100 mM; pH 7.5) containing 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and 1% SDS. Homogenates were diluted 1:26 and InsP levels were determined using the IP-One ELISA kit (Cisbio, Codolet, France) according to the manufacturer’s instructions. The average intra- and inter-assay coefficients of variation were 4.68 and 4.83%, respectively.

Accumulated inositol monophosphate (InsP1), reflecting the produced inositol triphosphate (InsP3) upon platelet stimulation, was measured by using the IP-One ELISA kit (Cisbio, Codolet, France) in the presence of LiCl (1 mM), which prevents the degradation of InsP1 into myo-inositol. Washed platelets (3 × 10⁸ platelets/mL) were stimulated with convulxin or EB in the absence or presence of effectors at 37 °C under stirring conditions, lysed after 5 min of activation, and centrifuged at 16,000× g for 10 min at 4 °C. InsP1–HRP conjugate and anti-InsP1 monoclonal antibody were pre-incubated with platelet lysates for 3 h prior to measurement, and InsP1 was quantified according to the manufacturer’s instructions.

Twelve male WT C57BL/6 mice (age: 8 weeks; Taconic) were injected with LiCl (10 mmoles/kg, s.c.) prior to the beginning of the dark cycle (6 pm). Six mice had free access to food, while the remaining six mice were fasted overnight. On the next morning (8 am), the mice were euthanized, and adipose depots (iWAT and eWAT) were isolated and quickly frozen on dry ice. Adipose tissues were homogenized in RIPA buffer, supplemented with LiCl (50 mM) and complete EDTA-free protease inhibitor cocktail (Roche). Briefly, 100–200 mg tissue was incubated in RIPA buffer with LiCl (50 mM) for 15 min on ice, followed by homogenization in lysing matrix E tubes using a Precellys® 24 homogenizer. The resultant supernatant was subjected to centrifugation (6000 x g) for 15 min to separate the ‘fat cake’ (white lipid layer) from the supernatant containing the loose pellet. The supernatant and the loose pellet (cell debris) were subjected to an additional centrifugation step (1200 x g, 15 min) to yield the protein lysate (supernatant). IP₁ levels in the protein lysates were measured using the IP-one ELISA kit from Cisbio following the manufacturer’s instructions.

Frozen tissue was weighed and homogenized by sonication in 10 μl/mg of tissue of Tris-HCl buffer (100 mM; pH 7.5) containing 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% SDS. Homogenates were diluted 1:50 and InsP levels were assessed with the IP-One ELISA kit (Cisbio, Codolet, France) according to the manufacturer’s instructions. Mean intra-assay and inter-assay coefficients of variation were 4.68 and 4.83%, respectively.

For the assessment of mGlu5 receptor signaling, mice were injected systemically with lithium chloride (10 mM, 100 μl, i.p.) followed, after 1 hour, by i.p. injection of a positive allosteric modulator for mGlu5 (VU0360172, 10 mg/kg i.p.) to activate mGlu5 receptors. Control mice were treated with lithium chloride followed by vehicle. Mice were sacrificed 60 min after the last injection and the brain regions were microdissected with a Vibratome (Leica Biosystems, Buccinasco, MI, Italy). The selected brain regions were weighed and homogenized by sonication in 10 μl/mg of tissue of Tris-HCl buffer (100 mM; pH 7.5) containing 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% SDS. Homogenates were diluted 1:50 and InsP levels were assessed with the IP-One ELISA kit (Cisbio, Codolet, France) according to the manufacturer’s instructions. Microplate manager 6 software (MPM6 Biorad) was used for plate reading.

All chemicals were obtained from Sigma Aldrich unless otherwise indicated. METx (Fig. 2) was designed in house and custom synthesised by Thermo Fisher Scientific, UK. Nanomerics’ Molecular Envelope Technology (N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan, GCPQ, Mw = 7.12 kDa, Mw/ Mn = 1.004, Mole% palmitoylation = 25%, Mole% quaternary ammonium groups = 12%) was obtained from Nanomerics Ltd. TrypLE™ Express was obtained from Thermo Fisher Scientific, UK. The IP-One ELISA kit was obtained from Cisbio Bioassays, USA and the cAMP Biotrak enzyme immunoassay kit was obtained from GE Life Sciences, USA. A7r5 rat smooth muscle cells and Madin-Darby Canine kidney (MDCK) cells were obtained from European Collection of Cell Cultures (ECACC). Male Wistar rats were obtained from Charles River (Charles River, UK) while Male Sprague Dawley rats were obtained from Harlan (Harlan, UK).