Pd10 sephadex g 25m column

BER liposomes were obtained by the thin-film hydration method [27 (link)]. Briefly, different stock solutions of PC (600 mg/mL), Chol (50 mg/mL), TS (50 mg/mL), DDAB (50 mg/mL) and DP (50 mg/mL) were dissolved in an organic phase composed of chloroform and methanol (9:1, v/v). Three different types of liposomes were prepared: PC, Chol and either TS, DDAB or DP were mixed at a molar ratio of 75:40:5 mM, respectively, in a round-bottomed flask. For BER containing liposomes, 5 mg of BER dissolved in 400 μL of methanol were mixed with the three lipids before the film formation. Organic solvents were evaporated in a rotary evaporator R-300 (Buchi, Flawil, Switzerland) at 40 °C for approximately 20 min, to form a film. The film was finally hydrated with 3 mL of PBS PH 7.4 during 30 min at 40 °C in a rotary evaporator without vacuum. Then, obtained liposomes were sonicated in a MicrosonTM XL 2000 ultrasonic cell disruptor (Misonix, Farmingdale, NY, USA) for 3 min at 8 Watts and stored at 4 °C overnight. To remove the free BER, BER precipitated overnight was discarded and BER liposomes were passed through a PD10 Sephadex^® G-25M column (GE Healthcare, Amersham, UK) using PBS as elution buffer. Finally, liposomes, named as PC:Chol:TS, PC:Chol:DDAB or PC:Chol:DP, were lyophilized with trehalose (10% w/v) and stored at 4 °C until further use.

Binding experiments of (SST14)₂-HSA and (SST28)₂-HSA for SSTRs were performed with HEK 293 cells stably expressing SSTR1-5 respectively. Labeling of Tyr¹-somatostatin with ¹²⁵I was performed using the chloramine-T method [18] (link). PD-10 Sephadex G-25M column (GE Healthcare) were used for further purification of the labeled tracer. HEK 293 cells stably expressing SSTR1-5 were seeded in 24-well plates at a density of 1.5×10⁵/well. 12 hours later, the growth medium was replaced with 0.2% bovine serum albumin in DMEM and cells were continuously incubated for another 2 hours at 37°C. Then, the medium was aspirated, and 0.25 ml of binding medium containing 50 pM ¹²⁵I-Tyr¹-somatostatin alone or with increasing concentrations of unlabeled (SST14)₂-HSA or (SST28)₂-HSA was added to the cells and incubated at 25°C for 1 hour. After that, the cells were washed twice with 0.5 ml of ice-cold phosphate-buffered saline to remove nonspecifically bound tracer. 0.5 ml of 1 N NaOH was added to each well and incubated at room temperature to solubilize the cells. Radioactivity was measured in a Perkin Elmer 1470 automatic γ counter [19] (link), [20] (link).