Uptake of [3H] 2-Deoxy-D-glucose was determined in THP-1 macrophages. Following indicated treatments, cells were washed twice in PBS and thereafter incubated for 7 minutes at 37°C with glucose-free media supplemented with 10 mM 2-Deoxy-D-glucose (D3179, Sigma-Aldrich) and 2.5 µCi/mL [3H] 2-Deoxy-D-glucose (NET549001MC, PerkinElmer). Assays were terminated by washing cells two times with ice-cold PBS. Subsequently, cells were solubilized on ice with cold lysis buffer containing 0.1% SDS, cOmplete™, EDTA-free Protease Inhibitor Cocktail (11873580001, Roche) and 70 units/mL Benzonase® Nuclease (E1014, Millipore). 3H was detected in 4 mL of Ultima Gold™ liquid scintillation cocktail (6013326, PerkinElmer) using a Hidex 300 SL scintillation counter and protein concentrations were determined using Pierce™ BCA Protein Assay Kit (23225, Thermo Scientific). Counts from each well were normalized by protein content.
Ultima gold liquid
Manufactured by PerkinElmer
ULTIMA GOLD liquid is a scintillation cocktail used in liquid scintillation counting (LSC) applications. It is a high-efficiency, low-background scintillation liquid designed for the measurement of radioactive samples. The product provides consistent performance and reliability for a wide range of sample types.
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Lab products found in correlation
3 protocols using ultima gold liquid
1
Glucose uptake in THP-1 macrophages
2
Quantification of OligoGM1 Internalization
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The possible internalization of OligoGM1 in hBLECs was determined by measuring the radioactivity associated to the cells. Briefly, at the end of direct transport, apical and basolateral solutions were collected, hBLEC filters were washed with RH buffer, and cells were detached by trypsin and lysed with 1mM Na3VO4, 1 mM PMSF, 2% (v/v) aprotinin, and 1% (v/v) IP in cold PBS. At this point, the lysed cell solution was counted to measure the radioactivity. All the cell lysates were combined with 5 mL of ULTIMA GOLD liquid (PerkinElmer), shaken, and counted for 20 min by liquid scintillation analyzer (TRI-CARB 2100TR, Packard). The resulting dpm radioactivity measures were used to estimate the molecule quantity.
3
GM1 and OligoGM1 Transport Experiments
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Transport experiments were carried out by adding tritium-labeled GM1 or OligoGM1 (1 × 106 dpm) to cold GM1 or OligoGM1, respectively, at different concentrations, as specified below.
To identify and quantify the GM1 or OligoGM1 molecules in every transport experiment, aliquots from apical A (5 μL) and basolateral B (200 μL) compartments were collected at indicated time points and combined with 5 mL of ULTIMA GOLD liquid (PerkinElmer), shaken, and counted for 20 min by liquid scintillation analyzer (TRI-CARB 2100TR, Packard). Resulting radioactivity measures were used to estimate the quantity of the molecules. Experiments were performed on an agitation plate at 37 °C (150 rpm, Heidolph Titramax 100) in a humidify atmosphere of 95% air/5% CO2. At least three inserts with cells were tested in each transport experiment for each condition.
To identify and quantify the GM1 or OligoGM1 molecules in every transport experiment, aliquots from apical A (5 μL) and basolateral B (200 μL) compartments were collected at indicated time points and combined with 5 mL of ULTIMA GOLD liquid (PerkinElmer), shaken, and counted for 20 min by liquid scintillation analyzer (TRI-CARB 2100TR, Packard). Resulting radioactivity measures were used to estimate the quantity of the molecules. Experiments were performed on an agitation plate at 37 °C (150 rpm, Heidolph Titramax 100) in a humidify atmosphere of 95% air/5% CO2. At least three inserts with cells were tested in each transport experiment for each condition.
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