Tri carb 2910tr liquid

For measurement of in vitro fat oxidation cells were incubated with ¹⁴C- labeled palmitate (250 µCi/ml; PerkinElmer, Boston, MA, USA) and non-labeled palmitate. Palmitate was coupled to fatty acid free BSA (7.5%) together with HEPES (100 mmol/L) and carnitine (40 mmol/L).The label solution contained ¹⁴C-labeled palmitate (1 μCi/ml), 100 μM non-labeled (cold) palmitate, 0.25% BSA, 12.5 mM HEPES, and 1 mM L-Carnitine. After SA incubation, complete (¹⁴CO₂) and incomplete (¹⁴ASM, acid soluble metabolites) oxidation products were measured, as previously described (36 (link)).
¹⁴ASM include acetyl-CoA, acetyl carnitines, ketone bodies, and TCA cycle intermediaries. Briefly, following incubation, medium was transferred to a custom-made Teflon 24-well CO₂ trapping plate that was sealed, 70% perchloric acid was injected (Hamilton syringe, 1705N) through a silicon layer in the lid directly into the media. This moved the CO₂ through a tunnel to an adjacent well where it was trapped in 1 N NaOH. After overnight trapping, complete and incomplete oxidation products were measured by Scintillation counting (using a Tri-Carb 2910 TR liquid scintillation analyzer, Perkin Elmer). For measuring endogenous fat oxidation, cells were pre-incubated for 24 h with ¹⁴C- labeled palmitate (250 µCi/ml; PerkinElmer, Boston, MA, USA) without carnitine and washed prior to SA incubations.

Membrane fractions isolated from Sf9 insect cells expressing either hBLT1-WT or hBLT1 mutants were incubated at RT for 2 h with [³H]–LTB4 (ARC) in assay buffer (10 mM Tris-HCl pH 7.4, 10 mM CaCl₂, 10 mM MgCl₂) in a total assay volume of 100 µL. The unbound ligand was removed by rapid filtration through GF/C glass fiber filters and 3 × 3 mL washes with 40 mM HEPES pH 7.4 and 0.2 % (w/v) CHAPS. Bound radioactivity was measured through liquid scintillation using Eco-Lume Liquid Scintillation Cocktail (MP Biomedicals) and detected using a Tri-Carb 2910TR liquid scintillation counter (Perkin Elmer). Competition studies were carried out by incubating membranes (20 μg total protein per well) with a range of concentrations of LTB4 and MK-D-046 (30 µM–0.007 nM) and with [³H]–LTB4 at 4 nM.

The production of superoxide was measured in the 15–20 mg fresh samples of the tissues using lucigenin (50 µmol/L)-enhanced chemiluminescence using a TriCarb 2910TR liquid scintillation analyser (TriCarb, Perkin Elmer, Waltham, MA, USA), as described previously by Kluknavsky et al. [26 (link)]. The results are expressed in the form of cpm/mg of wet tissue.