Glucose uptake in primary adipocytes was determined as previously described (Gliemann et al., 1984 (link)). Briefly, cells (7.5% [vol/vol] suspension) were incubated with or without insulin (concentrations shown in Figure Legends) in KRBH buffer in triplicates for 30 min, followed by the addition of D-14C(U)-glucose (2.5 µl/ml, NEC042, Perkin Elmer, Waltham, USA), and an additional 30 min of incubation. The uptake was terminated by spinning 300 µl of each cell suspension in microtubes containing 80 µl dinonylphtalate oil. The cell fraction was collected, dissolved in scintillation fluid (Optima Gold, Perkin Elmer), and subjected to scintillation counting. Glucose uptake in 3T3-L1 adipocytes was performed as outlined (Roccisana et al., 2013 (link)).
Nec042
Manufactured by PerkinElmer
Sourced in United States
The NEC042 is a laboratory instrument designed for sample analysis. It provides accurate and reliable measurement capabilities. The core function of the NEC042 is to perform sample analysis tasks within a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Optima gold, by PerkinElmer (3 mentions)
Ultima gold, by PerkinElmer (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Bodipy, by Thermo Fisher Scientific (1 mentions)
Glut1 antibody, by Santa Cruz Biotechnology (1 mentions)
Rapamycin, by Enzo Life Sciences (1 mentions)
Chloroquine, by Enzo Life Sciences (1 mentions)
5 protocols using nec042
1
Glucose Uptake in Adipocytes
2
Measuring Glucose Uptake in Primary Adipose Cells
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Primary adipose cells were isolated from epididymal adipose tissue as previously described (Rodbell, 1964 (link)). The isolated cells were suspended (5% suspension) in Krebs-Ringer (KRH) medium containing 25 mM HEPES pH 7.4, 200 nM adenosine, 2 mM glucose and 3% BSA (w/v), and glucose uptake measured as previously described (Gliemann et al., 1984 (link)). Briefly, cells were incubated in KRH medium (37 °C, shaking water bath) in triplicates with or without insulin (28 nM) for 30 min, followed by addition of d -14C(U)-glucose (2.5 μl/ml, NEC042, Perkin Elmer), and incubated for 30 min. The uptake was terminated by spinning 300 μl of each cell suspension in microtubes containing 80 μl dinonylphtalate oil. The cell fraction was collected, dissolved in scintillation fluid (Optima Gold, Perkin Elmer, Upplands Väsby, Sweden) and subjected to scintillation counting.
3
Insulin-Stimulated Glucose Uptake Assay
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Glucose uptake was determined as previously described (Gliemann et al., 1984 (link)). Cells (7.5 % (v/v) suspension) were incubated with or without 10 nM insulin in KRBH buffer in triplicates for 30 min, followed by the addition of D-14C(U)-glucose (2.5 μL/ml, NEC042, Perkin Elmer, Waltham, United States), and an additional 30 min of incubation. The uptake was terminated by centrifugation of 300 µL of each cell suspension in microtubes containing 80 µL dinonylphtalate oil. The cell fraction was collected, dissolved in scintillation fluid (Ultima Gold, Perkin Elmer), and subjected to scintillation counting.
4
Antibodies and Reagents for Cell Signaling
5
Glucose Uptake Measurement Protocol
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Glucose uptake was determined as previously described50 (link). Cells were incubated in KRBH medium without glucose in triplicate without or with insulin (0.01 or 10 nM) for 30 min., followed by addition of D-14C(U)-glucose (2.5 µl/ml, NEC042, Perkin Elmer), and an additional 30 min incubation. The uptake was terminated by spinning 300 µl of each cell suspension in microtubes containing 80 µl dinonylphtalate oil. The cell fraction was collected, dissolved in scintillation fluid (Optima Gold, Perkin Elmer) and subjected to scintillation counting.
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