Villus cells were isolated from the ileum of the experimental animals by a calcium chelation technique as previously described [19 (link)] with few modifications. Briefly, isolated distal ileal villus cells were washed twice in TMA-HEPES buffer (50 mM KCl, 0.1 mM MgSO4, 50 mM Mannitol, 50 mM HEPES-Tris (pH 7.5), and 100 mM TMA-Cl) and suspended in 100 µL of the same buffer. Next, 10 μL of the cells from the suspension were added to 100 μL of reaction media containing 0.1 mM taurocholate (TCA; Sigma-Aldrich Corporation, St. Louis, MO, USA) with 100 μM 3H-taurocholate (PerkinElmer, Inc., Waltham, MA, USA), 50 mM KCl, 0.1 mM MgSO4, 50mM Mannitol, 50 mM HEPES-Tris (pH 7.5), and either 100 mM TMA-Cl or 100 mM NaCl. The reaction was stopped at two minutes by adding 1 mL of ice-cold TMA-HEPES buffer. The stopped reaction mixture was filtered on 0.65 μm Millipore (HAWP) filter and washed twice with ice-cold stop solution. The reactions were carried out in triplicate for each of the two reaction mixtures. The filter was dissolved in 5 mL of scintillation fluid (Ecoscint A, National Diagnostics), and radioactivity was determined in a Beckman 6500 scintillation counter (Beckman Coulter Inc., Brea, CA, USA).
Beckman 6500 scintillation counter
Manufactured by Beckman Coulter
Sourced in United States
The Beckman 6500 scintillation counter is a highly sensitive instrument used for the detection and quantification of radioactive samples. It utilizes the principles of liquid scintillation counting to measure the level of radioactivity in a sample. The core function of the Beckman 6500 is to accurately detect and measure the energy and intensity of ionizing radiation emitted by the sample.
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2 protocols using beckman 6500 scintillation counter
1
Isolation and Characterization of Ileal Villus Cells
2
Radioimmunoassay for Corticosterone Quantification
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A radioimmunoassay for corticosterone was used to quantify hormone levels (34) . Briefly, plasma volumes were measured (30 ll) and equilibrated with 2000 c.p.m. of tritiated corticosterone to determine recoveries. Steroids were extracted, for approximately 3 h, using freshly redistilled dicholoromethane, dried under nitrogen at 35 °C, and reconstituted in 550 ll of phosphatebuffered saline with gelatine. The next day, 200 ll was placed in duplicate assay tubes, and 100 ll was used to determine the percent recovery. Each assay tube received 100 ll (approximately 10 000 c.p.m.) of tritiated corticosterone (Perkin Elmer, Boston, MA, USA) and 100 ll of antiserum (B3-163; Esoterix Inc., Austin, TX, USA). Unbound steroid was stripped using 500 ll of dextran-coated charcoal followed by centrifugation. The supernatant was decanted, combined with scintillation fluid (Ultima Gold; Perkin Elmer), and then counted for 10 min or within 2% accuracy on a Beckman 6500 scintillation counter (Beckman Coulter, Fullerton, CA, USA). Mean recovery for corticosterone was 90%. The mean detection limit for the assay was 0.8 ng/ml. All basal samples for the hybridisation study were run within the same assay with an intra-assay variation of 10.28%. Stress series samples were run across multiple assays with an inter-and intra-assay variation of 8.94 and 9.07, respectively.
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