Filtermat harvester

The radiolabeled precursor ³H-thymidine is incorporated into DNA strands during cell replication and radioisotope incorporation is often used as a measure of cell proliferation. All cells were pulsed with 20 μl of RPMI 1640 media containing ³H-thymidine (0.5 μCi) per well and incubated in 5% CO2 at 37 °C for a minimum of 8 h s before harvesting. The cells were then harvested by a Filtermat Harvester (Perkin Elmer) onto Glass fiber matt filters (part No. 6005422) that captures DNA. The filters were then air-dried and placed in Omni Filter cassette and 30 μl of MicroScintTM (Perkin Elmer Cat No 6013611) liquid added, then sealed with TopSeal (Perkin Elmer, Part No 6050195) and loaded on TopCount NXT reader (Perkin Elmer) for determination of incorporation of ³H-thymidine.

Two micrograms of human GhrR membranes (prepared from HEK293 cells stably expressing the fusion protein hGhrR-eYFP), 60 pM [¹²⁵I-His⁹]ghrelin (PerkinElmer, Waltham, MA, USA), and different concentrations of the test compound were incubated in HEPES buffer (20 mM HEPES, 20 mM CaCl₂, 0.8 mM MgCl₂, 1% Pefabloc, 1% bovine serum albumin, pH 7.4) in a 96-well plate on a shaker (200 rpm) for 2.5 h at rt. Each concentration was tested in duplicate. The membrane-bound radioligand was trapped on a Filtermat B (PerkinElmer) pre-soaked with 0.1% polyethylenimine in H₂O by using a filtermat harvester (PerkinElmer). After drying for 20 min at 55 °C, a Meltilex scintillation sheet (PerkinElmer) was melted onto the filtermat and the samples were counted in a Microbeta scintillation counter (PerkinElmer). For each compound, all data points (n = 4–6) from at least two independent experiments were combined and IC₅₀ values were obtained by a sigmoidal dose-response fit. Data were normalized to no competition (100%, minimal ghrelin effect) and full competition (0%, maximal ghrelin effect).