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Net328a250uc

Manufactured by PerkinElmer
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The NET328A250UC is a laboratory equipment device. It serves as a core function without further interpretation or extrapolation on its intended use.

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Lab products found in correlation

Net328250uc, by PerkinElmer (2 mentions) Fibronectin, by Merck Group (1 mentions) Cytochalasin b, by Merck Group (1 mentions) D8375, by Merck Group (1 mentions) C6762, by Merck Group (1 mentions) I0516, by Merck Group (1 mentions) Mgcl2, by Merck Group (1 mentions) Cacl2, by Merck Group (1 mentions) Hgcl2, by Merck Group (1 mentions)

5 protocols using net328a250uc

1

Insulin-Stimulated Glucose Uptake Assay

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The THRSP-silenced or overexpressing SGBS adipocytes were washed carefully with PBS and starved in glucose- and serum-free DMEM for 24 h. The cells were treated with 100 nM insulin for 20 min and then incubated with 50 nM deoxy-d-glucose and [3H] deoxy-d-glucose (0.5 µCi/well; Perkin Elmer; NET328A250UC) for 5 min. Glucose uptake was terminated by three washes with ice-cold PBS. The cells were lysed with 0.1% SDS and radioactivity was measured by liquid scintillation counting.
Ahonen M.A., Höring M., Nguyen V.D., Qadri S., Taskinen J.H., Nagaraj M., Wabitsch M., Fischer-Posovszky P., Zhou Y., Liebisch G., Haridas P.A., Yki-Järvinen H, & Olkkonen V.M. (2022). Insulin-inducible THRSP maintains mitochondrial function and regulates sphingolipid metabolism in human adipocytes. Molecular Medicine, 28, 68.
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2

Hippocampal Glucose Uptake Assay

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Hippocampal slices were washed with buffer (15 mM HEPES (cat: H3375), 135 mM NaCl (cat: s3014), 5 mM KCl (cat: P5405), 1.8 mM CaCl2 (cat: C1016), and 0.8 mM MgCl2 (cat: 208337) supplemented with 0.5 mM glucose (Cisternas et al., 2014a (link)). Then, the slices were incubated for 0–90 min with 1–1.2 μCi 2-[1,2-3H(N)]-deoxy-D-glucose (cat: NET328250UC, PerkinElmer, United States) or 3H-2-deoxyglucose (cat: NET328A250UC, PerkinElmer, United States) at a final specific activity of 1–3 disintegrations/min/pmol (∼1 mCi/mmol). Glucose uptake was arrested by washing the cells with ice-cold PBS supplemented with 1 mM HgCl2 (cat: 203777, Sigma-Aldrich, United States). The incorporated radioactivity was quantified by liquid scintillation counting.
Cisternas P., Oliva C.A., Torres V.I., Barrera D.P, & Inestrosa N.C. (2019). Presymptomatic Treatment With Andrographolide Improves Brain Metabolic Markers and Cognitive Behavior in a Model of Early-Onset Alzheimer’s Disease. Frontiers in Cellular Neuroscience, 13, 295.
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3

Insulin-Stimulated Glucose Uptake Assay

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C2C12 cells were plated on a 6-well plate coated with 5 ug/ml fibronectin (f1141; Sigma-Aldrich). 42 h after transfection (with Lipofectamine 2000), cells were washed in warm PBS, starved in DMEM/0.1% BSA for 6 h, and washed again. After treatment with 200 nM insulin/PBS (I0516; Sigma-Aldrich) for 30 min at 37°C, cells were washed and incubated for 10 min at 37°C with 1 uCi/ml 2-deoxy-d-[3H]glucose (NET328A250UC; PerkinElmer) and 0.1 mM cold 2-deoxy-d-glucose (D8375; Sigma-Aldrich). 20 nM of cytochalasin B (glucose transport inhibitor; C6762; Sigma-Aldrich) was added to control wells. Then, cells were washed in cold PBS, lysed in 0.2 N NaOH for 2 h at room temperature, and radioactivity was determined using scintillation fluid.
Cohen S., Lee D., Zhai B., Gygi S.P, & Goldberg A.L. (2014). Trim32 reduces PI3K–Akt–FoxO signaling in muscle atrophy by promoting plakoglobin–PI3K dissociation. The Journal of Cell Biology, 204(5), 747-758.
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4

Glucose Uptake Assay in Myotubes

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The assay of glucose uptake was modified from previously described protocol [26] (link). Serum starvation was performed in HBSS (5 mM glucose) for at least 4 h to further enhance the basal and insulin-induced glucose uptake. After starvation, the culture medium of myotubes in 6-well plates were replaced with Krebs-Ringer phosphate (KRPH) buffer containing 0.2 μCi 2-deoxy-D-[3 H]-glucose (2-DG; NET328A250UC, Perkin Elmer) with/without 100 nM insulin and incubate at 37 °C for another 2 h. One well was treated with 10 μM cytochalasin B (CB) for inhibiting actin cytoskeleton mediated glucose uptake and thus served as non-specific/negative glucose uptake control. After two times wash with cold 1x phosphate-buffered saline (PBS), cells were lysed with 500 μl radioimmunoprecipitation assay (RIPA) buffer. The radioactivity in the lysate is quantified by liquid scintillation counting. The value of the result is normalized to protein amount and showed as cpm/μg.
Chang H.C., Kao C.H., Chung S.Y., Chen W.C., Aninda L.P., Chen Y.H., Juan Y.A, & Chen S.L. (2018). Bhlhe40 differentially regulates the function and number of peroxisomes and mitochondria in myogenic cells. Redox Biology, 20, 321-333.
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5

Quantification of Glucose Uptake in Cells

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After activator/inhibitor treatments, cells were washed with incubation buffer (15 mM HEPES (cat: H3375), 135 mM NaCl (cat: s3014), 5 mM KCl (cat: P5405), 1.8 mM CaCl2 (cat: C1016), and 0.8 mM MgCl2 (cat: 208337), all the reagents were purchased from Sigma-Aldrich, USA) supplemented with 0.5 mM glucose (Cisternas et al. 2014a). Cells were then incubated for 15 s with 1–1.2 μCi 2-deoxy-D-[1,2-(N)3H] (cat: NET328250UC, PerkinElmer, USA) or glucose ([2-3H]-DG (cat: NET328A250UC, PerkinElmer, USA) at a final specific activity of 1–3 disintegrations/min/pmol (~1 mCi/mmol). Glucose uptake was arrested by washing the cells with ice-cold PBS supplemented with 1 mM HgCl2 (cat: 203777, Sigma-Aldrich, USA). The incorporated radioactivity was quantified by liquid scintillation counting. In separate experiments, brain slices were incubated with different drugs for 1 h, then treated with [2-3H]-DG for 15 min and processed as described above. The kinetic parameters were determined using a single rectangular hyperbola of the form 𝑉max ∗ [glc]/(𝐾𝑚 + [glc]), adjusted to the data by nonlinear regression using SigmaPlot 12 (Barros et al. 2009 (link)).
Cisternas P., Zolezzi J.M., Martinez M., Torres V.I., Wong G.W, & Inestrosa N.C. (2018). Wnt-induced activation of glucose metabolism mediates the in vivo neuroprotective roles of Wnt signaling in Alzheimer disease. Journal of neurochemistry, 149(1), 54-72.
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