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2-DOG is a laboratory reagent used in the study of cellular metabolism. It is a glucose analog that inhibits the enzyme hexokinase, which is involved in the first step of glycolysis. 2-DOG can be used to measure glucose uptake and metabolism in cells.

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Lab products found in correlation

D5030 10x1l, by Merck Group (2 mentions) Ls6500 scintillation counter, by Beckman Coulter (2 mentions) Calf serum, by Merck Group (1 mentions) Salubrinal, by R&D Systems (1 mentions) Tumor necrosis factor (tnf), by R&D Systems (1 mentions) Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions) Fetal bovine serum (fbs), by Merck Group (1 mentions) Thapsigargin, by Merck Group (1 mentions)

6 protocols using 2 dog

1

2-DOG Lifespan Assay in C. elegans

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For performing 2-DOG lifespan, NGM agar was supplemented with 2-DOG (Sigma-Aldrich, D8375) to a final concentration of 5 mM. Synchronized egg population (100–150) obtained by sodium hypochlorite treatment of gravid worms grown on E. coli OP50 was transferred to control RNAi plates without 2-DOG. At young adult (YA) stage, approximately 50 worms were transferred to the 2-DOG-containing or control plates, both overlaid with FUDR, in three technical replicates. Worms were then scored as live or dead every alternate day. Sick worms with ruptured vulva rupture were censored from the population.
Chamoli M., Goyala A., Tabrez S.S., Siddiqui A.A., Singh A., Antebi A., Lithgow G.J., Watts J.L, & Mukhopadhyay A. (2020). Polyunsaturated fatty acids and p38-MAPK link metabolic reprogramming to cytoprotective gene expression during dietary restriction. Nature Communications, 11, 4865.
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2

2-DOG Life Span Assay in C. elegans

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Plates for performing 2-DOG life span were prepared from the same batch of NGM agar as the control plates except that the 2-DOG (Sigma-Aldrich, D8375) was added in the media to a final concentration of 5 mM from a sterile 0.5 M stock solution made in water. Synchronized egg population (100–150) obtained by sodium hypochlorite treatment of gravid worms grown on E. coli OP50, was exposed to control plates (without 2- DOG). At YA stage, approximately 50 worms were transferred to the plates containing 2-DOG and plates without 2-DOG, overlaid with FUDR, in three technical replicates. Worms were scored every alternate day as mentioned above.
Goyala A., Baruah A, & Mukhopadhyay A. (2020). The genetic paradigms of dietary restriction fail to extend life span in cep-1(gk138) mutant of C. elegans p53 due to possible background mutations. PLoS ONE, 15(11), e0241478.
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3

2-DOG Lifespan Assay in C. elegans

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Plates for performing 2-DOG life span were prepared from the same batch of NGM agar as the control plates except that the 2-DOG (Sigma-Aldrich, D8375) was added in the media to a final concentration of 5 mM from a sterile 0.5 M stock solution made in water. Synchronized egg population (100-150) obtained by sodium hypochlorite treatment of gravid worms grown on E. coli OP50, was exposed to control plates (without 2-DOG). At YA stage, approximately 50 worms were transferred to the plates containing 2-DOG and plates without 2-DOG, overlaid with FUDR, in three technical replicates. Worms were scored every alternate day as mentioned above.
Goyala A., Baruah A., & Mukhopadhyay A. (2020). The genetic paradigms of dietary restriction fail to extend life span in cep-1(gk138) mutant of C. elegans p53 due to possible background mutations.
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4

Murine 3T3-L1 Adipocyte Differentiation

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Murine 3T3-L1 pre-adipocytes, obtained from Dr. Green’s laboratory (7 (link)), were grown, maintained and induced to differentiate using a standard protocol (8 (link)). Fully differentiated adipocytes were maintained in DMEM (Sigma-Aldrich, St Louis, MO) supplemented with 10% FBS (Sigma-Aldrich) until two days before experimentation when cells were fed with 10% calf serum (Sigma-Aldrich). Prior to treatments media was changed to low-glucose (5.5 mM) DMEM (Sigma-Aldrich) and 1% calf serum overnight. For 20 hr treatments, cells were treated with 2-DOG and LPS or TNF concurrently. For shorter treatments, cells were pretreated with 2-DOG (Sigma-Aldrich), thapsigargin (Sigma-Aldrich) or salubrinal (R&D Systems, Minneapolis, MN) for 30 min and then treated with TNF (R&D Systems) for 1–4 hrs.
Grant R.W., Boudreaux J.I, & Stephens J.M. (2016). 2-deoxyglucose inhibits induction of chemokine expression in 3T3-L1 adipocytes and adipose tissue explants. Obesity (Silver Spring, Md.), 25(1), 76-84.
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5

Glucose Uptake Assay in 3T3-F442A Cells

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3T3-F442A cells incubated in 24-well culture dishes were washed three times with serum free DMEM, then serum starved for 16–18 hours in 5 mM Glucose DMEM and 1% BSA. Cells were then washed two times with PBS and incubated in glucose free and pyruvate rich (1 mM) DMEM (Sigma D5030-10X1L) for 30 minutes at 37°C with 5% CO2. Cells were then treated with or without cytochalasin B (30μM final, in DMSO Sigma C6762-10mg). H3 2-Deoxy-D-Glucose (2-DOG) (NET 328A001 mL, Perkin Elmer) was diluted 1:40 in H1 (link) 2-DOG (Sigma D6134-5g) then added to cells at a final concentration of 10 mM 2-DOG. Insulin was then added at a final concentration of 100 nM and cells were incubated for 30 minutes at 37 °C with 5% CO2. Following incubations cells were washed three times with PBS, and suspended in 500 μL 1M NaOH by pipetting up and down. Cell lysates were then freeze thawed twice diluted in Ultima Gold scintillation fluid and analyzed using Beckman Coulter LS-6500 scintillation counter. Protein concentrations were assessed in parallel using BCA analysis. H3 Content was then normalized to H3 2-DOG standard curves and glucose uptake values were generated normalized to protein content.
Zhang Y., Xie L., Gunasekar S.K., Tong D., Mishra A., Gibson W.J., Wang C., Fidler T., Marthaler B., Klingelhutz A., Abel E.D., Samuel I., Smith J.K., Cao L, & Sah R. (2017). SWELL1 is a regulator of adipocyte size, insulin signaling and glucose homeostasis. Nature cell biology, 19(5), 504-517.
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6

Glucose Uptake Assay in 3T3-F442A Cells

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3T3-F442A cells incubated in 24-well culture dishes were washed three times with serum free DMEM, then serum starved for 16–18 hours in 5 mM Glucose DMEM and 1% BSA. Cells were then washed two times with PBS and incubated in glucose free and pyruvate rich (1 mM) DMEM (Sigma D5030-10X1L) for 30 minutes at 37°C with 5% CO2. Cells were then treated with or without cytochalasin B (30μM final, in DMSO Sigma C6762-10mg). H3 2-Deoxy-D-Glucose (2-DOG) (NET 328A001 mL, Perkin Elmer) was diluted 1:40 in H1 (link) 2-DOG (Sigma D6134-5g) then added to cells at a final concentration of 10 mM 2-DOG. Insulin was then added at a final concentration of 100 nM and cells were incubated for 30 minutes at 37 °C with 5% CO2. Following incubations cells were washed three times with PBS, and suspended in 500 μL 1M NaOH by pipetting up and down. Cell lysates were then freeze thawed twice diluted in Ultima Gold scintillation fluid and analyzed using Beckman Coulter LS-6500 scintillation counter. Protein concentrations were assessed in parallel using BCA analysis. H3 Content was then normalized to H3 2-DOG standard curves and glucose uptake values were generated normalized to protein content.
Zhang Y., Xie L., Gunasekar S.K., Tong D., Mishra A., Gibson W.J., Wang C., Fidler T., Marthaler B., Klingelhutz A., Abel E.D., Samuel I., Smith J.K., Cao L, & Sah R. (2017). SWELL1 is a regulator of adipocyte size, insulin signaling and glucose homeostasis. Nature cell biology, 19(5), 504-517.
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