Wild-type (14028s) or mgtC (EL4) Salmonella were grown in MOPS medium containing 10 μM MgCl2 and 500 μM K2HPO4 during 3 h. Wild-type (14028s) or ∆3Pi (RB39) cells harboring either pVector or pMgtC were grown in MOPS containing 250 μM MgCl2 and 500 μM K2HPO4 until OD600 ∼0.2, at which point, MgtC expression was induced for 15 min with the addition of 750 μM IPTG. To assay the transport of Pi, 20 μCi of radioactive Pi solution (10 μL from a 2 mCi K2H32PO4 at a concentration of 2 mM K2H32PO4, PerkinElmer catalog number NEX055) was added to 1 mL cell suspension. At the indicated time points, 50 μL of each sample was submitted to rapid filtration through 0.45 μm mixed cellulose ester membrane filters (Whatman) with an applied vacuum. The filters were washed three times with 1 mL PBS buffer and subsequently soaked in 5 mL scintillation fluid (Research Products International). The amount of radioactivity taken up by the cells was determined with a scintillation counter (Triathler multilabel tester, HIDEX) using the 32P-window and by counting each vial for 20 s. Radioactive counts per minute were normalized by protein content using a Rapid Gold BCA Protein Assay Kit (Pierce). 32Pi uptake of each sample was normalized against the corresponding control in each independent experiment.
Scintillation fluid
Manufactured by Research Products International
Scintillation fluid is a liquid medium used in scintillation counting, a technique for detecting and measuring ionizing radiation. The fluid contains organic compounds that emit light when excited by radiation, allowing for the detection and quantification of radioactive samples.
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2 protocols using scintillation fluid
1
Phosphate Uptake Assay in Salmonella
2
Measuring β2-AR Abundance by Radioligand Binding
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The cell surface abundances of 2 AR WT and 2 AR Y219A were measured by whole-cell binding with the hydrophilic radioligand [ 3 H]-CGP 12177 (PerkinElmer). Human embryonic kidney-293 (HEK 293) cells were transiently transfected with pBK- 2 AR WT or pBK- 2 AR Y219A with Fugene 6 (Roche), and the cells were harvested 48 hours later by gentle agitation with 0.02% EDTA at 4°C. Cells were resuspended in assay buffer (10 mM Hepes in minimum essential medium) and incubated with 30 nM [ 3 H]-CGP 12177 for 3 hours on ice. Propranolol (10 M) was used to account for nonspecific binding. Unbound [ 3 H]-CGP 12177 was separated by filtration onto grade B glass fiber filters soaked in water with a 96-well-format Brandel harvester. The GF/B filters were then rapidly washed three times with ice-cold buffer [50 mM tris-HCl (pH 7.5), 12.5 mM MgCl 2 , and 2 mM EDTA (pH 8.0)] and soaked in scintillation fluid (Research Products International) overnight. Bound [ 3 H]-CGP 12177 was quantified by the Tri-Carb 2800TR Liquid Scintillation Analyzer (PerkinElmer). All data represent at least three independent experiments; SE and analysis were performed in GraphPad Prism.
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