Tri carb 2910tr low activity liquid scintillation analyzer

In experiment 1, after 6 weeks of treatment, TG-rich lipoprotein-like particles double-labeled with glycerol tri[³H]oleate (American Radiolabeled Chemicals, USA) and [¹⁴C]cholesteryl oleate (American Radiolabeled Chemicals, USA) were prepared as previously described⁴⁷ (link) and injected into the tail vein of the mice (1.0 mg TG in 200 μL saline per mouse). Blood samples were drawn from the tail vein at 2, 5, 10, and 15 min after the particle injection. Subsequently, mice were killed by CO₂ inhalation and perfused with ice-cold PBS before organs were collected. Plasma samples collected following the particle injection and tissue samples that had been dissolved overnight at 55°C in Solvable (PerkinElmer, The Netherlands) were mixed with Ultima Gold liquid scintillation cocktail (PerkinElmer, The Netherlands). ³H and ¹⁴C activity in the samples (disintegrations per minute; dpm) were quantified using a Tri-Carb 2910TR low-activity liquid scintillation analyzer (PerkinElmer, The Netherlands). Decay of ³H and ¹⁴C radioactivity in plasma was expressed as the percentage of injected radioactive dose. Uptake of ³H and ¹⁴C radioactivity by the organs was expressed as the percentage of injected radioactive dose per gram tissue.

Glycerol tri [³H]oleate ([³H]triolein [³H]TO; NET431L005MC, PerkinElmer)-labeled TG-rich lipoprotein (TRL)-like particles were prepared as previously described,²⁴ and [¹⁴C]deoxy-d-glucose ([¹⁴C]DG; EC495A250UC, PerkinElmer) was added to the emulsion (5:1 ³H:¹⁴C activity ratio). Directly after the collection of 4-h fasted blood, mice (cohort 1) were intraperitoneally injected with 2 g kg⁻¹d-glucose to induce a standardized fed state while avoiding the production of endogenous GIP and GLP1 by the intestine. Half an hour later, mice were intravenously injected with the mixture of [³H]TO-labeled TRL-like particles (1 mg TG per mouse) and [¹⁴C]DG in 200 μL PBS, and killed by CO₂ inhalation 15 min thereafter. After collecting blood via heart puncture to assess ALT activity (MAK052, Sigma–Aldrich), mice were perfused with ice-cold PBS. Collected tissues (max. 200 mg) were weighed and dissolved overnight at 55 °C in 500 μL Solvable (Perkin Elmer), after which 5 mL Ultima Gold liquid scintillation cocktail (PerkinElmer) was added to determine ³H and ¹⁴C activity using a Tri-Carb 2910 TR Low Activity Liquid Scintillation Analyzer (PerkinElmer). Uptake of [³H]TO- and [¹⁴C]DG-derived radioactivity by organs was expressed as the percentage of injected dose per gram of wet tissue.

On the day before treatment H9c2 or NRK-52E cells (40.000 per well) were seeded in a 24-well plate in standard culture medium. For treatment, the medium was exchanged for 200 µL of standard culture medium containing 0.25 µCi/mL methyl-³H-thymidine (PerkinElmer) and respective concentrations of remdesivir, 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (8-CPT-cAMP; Abcam) or solvent control (dimethylsulfoxide (dmso); Sigma). Cells were incubated for 6 h at 37 °C, 0% CO₂, followed by 3 washing steps with phosphate buffered saline (PBS), overnight incubation with 5% tricarboxylic acid (TCA; Applichem) and cell lysis with 0.5 mM NaOH (Applichem). Lysates were mixed with 4 mL Rotiszint^® eco plus (Roth) in 6.5 mL scintillation-tubes (Roth) and counts per minute were recorded by a Tri-Carb^® 2910 TR Low Activity Liquid Scintillation Analyzer (PerkinElmer). Each condition was measured in triplicates.