Tri carb 2910tr liquid scintillation counter

Chemical synthesis was performed following (Keenan and Kruczek, 1975 (link)). Obtained [³H]geranylgeranyl alcohol, specific activity 2.5 Ci mol⁻¹, was dissolved in hexane at a concentration of 3.7 × 10⁷ dpm μl⁻¹. In vivo labeling and extract preparation were performed as described in (Gutkowska et al., 2004 (link)) and proteins were separated by 15% SDS‐PAGE. Fractions corresponding to 20–30 kDa were cut from the gel, solubilized in 5.5% H₂O₂ at 65°C for 24 h, and measured for radioactivity in a Tri‐Carb 2910TR liquid scintillation counter (Perkin Elmer, Waltham, MA, USA) with Insta‐Gel Plus scintillation liquid (Packard, Waltham, MA, USA). Bands of the same molecular mass from the same gel but containing non‐labeled samples were treated as control. For gel autoradiography, SDS‐PAGE gels were soaked in salicylic acid as described in (Hala et al., 2010 (link)), dried under vacuum, and exposed on Kodak X‐Omat AR5 film at −70°C for 1 month.

The sodium (Na⁺)-dependent bile acid transport of taurocholic acid (TCA) was assessed by hASBT-MDCK cells and hNTCP-HEK293T cells. Cells were washed thrice with pre-warmed Hanks’ Balanced Salt Solution (HBSS) containing NaCl (8 g/l, 0.8%) or sodium-free solution with 137 mM TEA-Cl replacing NaCl. Cells were then incubated at 37°C for 5 or 10 min (periods of linear uptake for NTCP and ASBT, respectively) with 0–100 μM cold TCA spiked with 1 μCi/ml [³H]-TCA in HBSS or sodium-free buffer (SFB) donor solutions. Wells were quenched and then washed twice using ice cold sodium-free buffer (SFB). Cells were lysed with 300 μl ACN and left to evaporate at RT for 2–3 h. Lysates were resuspended in 1:1 ACN:H₂O and centrifuged at 12,000g for 10 min. Supernatants were counted for radioactivity using a Tri-Carb® 2910 TR liquid scintillation counter (PerkinElmer, Waltham, MA). TCA uptake rates were determined as pmol/surface area (cm²)/min. Results were fitted to a straight line for SFB samples or a modified Michaelis–Menten equation for HBSS samples; no weighting was used, and regression was performed using Prism (GraphPad, San Diego, CA). This modified equation [Eq. (1)] accounts for passive permeability,
$$V=\frac{V_{max}\left[S\right]}{K_m+\left[S\right]}+P_{pass}\left[S\right]$$
where V is TCA flux, [S] is TCA concentration and P_pass is passive TCA permeability. P_pass was determined on the same occasion by TCA uptake in SFB.

HEK cells were transfected with plasmid DNA. Seven hours post transfection, cells were seeded on p12 culture dishes previously coated overnight with poly-L-lysine. 24 hr post-transfection the cells were washed with Krebs-Ringer (KR) buffer (146 mM NaCl, 3 mM KCl, 1 mM CaCl₂, 10 mM KH₂PO₄/K₂HPO₄) at pH 7.4 and incubated for 15 min with KR buffer (pH 5.5) containing 100 μM of non-radiolabeled amino acids and 0.5 μCi of ³H labeled amino acids. The cells were washed with cold KR buffer (pH 7.4) on ice, lysed with 0.1 M NaOH and collected. 0.2 M KR buffer (pH 6.2) was added to the mix containing the lysed cells in order to neutralize the pH. The protein content of the cell lysates was analyzed and the remaining cell lysates were used for radioactive measurements. Briefly, the samples were mixed with Ultima GoldTM liquid scintillation cocktail (PerkinElmer) and the disintegrations per minute (DPM) were measured in a TriCarb 2910 TR liquid scintillation counter (PerkinElmer). The radioactivity measurements were corrected by the protein concentrations.