Rhodamine 6G (R6G) efflux assay was performed as described previously18 (link). Briefly, 108 young and old (10 gen) cells from isolate S1 or exponentially growing cells of all isolates were starved for 2 h in presence of 5 mM deoxy glucose (Sigma-Aldrich) at 37 °C with shaking and were washed three times with 1X PBS. R6G (Sigma-Aldrich) was then added to the washed cells at a final concentration of 10 µM and incubated for 30 mins at 37 °C with shaking to initiate R6G import. After incubation, the cells were washed three time in 1X PBS, and efflux was initiated by adding 2% glucose. Samples were collected every 30 mins, and the fluorescence of the supernatants were measured at excitation and emission wavelengths of 525 nm and 555 nm respectively. The assay was performed in triplicate.
Deoxyglucose
Manufactured by Merck Group
Sourced in United States
Deoxyglucose is a chemical compound used in laboratory settings as a tool for research and analysis. It is structurally similar to glucose, but with one of the hydroxyl groups replaced by a hydrogen atom. This modification affects the compound's biological properties and makes it useful for specific applications in the field of biochemistry and molecular biology.
Automatically generated - may contain errors
4 protocols using deoxyglucose
1
Rhodamine 6G Efflux Assay in Microbial Cells
2
Controlled Expression of Nuclear Membrane Proteins
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HeLa cells were maintained in DMEM containing 10% fetal bovine serum and penicillin/streptomycin at 37°C, 5% CO2. Transient transfections were performed using the X-tremeGene transfection reagent (Roche). Reporter constructs were stably integrated into the FRT site of a HeLa cell line containing a tetracycline repressor. Expression of the reporter proteins 2×RFPtevSUN2-GFP, 2×RFPtevSUN2(1–260)-GFP, 2×RFPtevLBR(fl)-GFP, and 2×RFPtevGFP-LBR(1–245)-GFP was induced for 16 h by the addition of 200 ng/ml tetracycline. Expression of 2×RFPtevLBR(1–245)-GFP and 2×RFPtevLAP2β-GFP was induced for 16 h by 20 ng/ml. AR proteins were induced for 8 h with 200 ng/ml tetracycline in HeLa cell lines constitutively expressing H2B-Plum-FKBP. The HeLa cell line (Mitchell et al., 2010 (link)) stably expressing rat POM121-3×GFP (Rabut et al., 2004 (link)) from the pEGFP backbone (CMV promoter; BD) has been described previously.
For depletion of cellular ATP (and GTP; Schwoebel et al., 2002 (link)), cells were treated with 6 mM deoxyglucose (Sigma-Aldrich) and 10 mM NaN3 in glucose-free DMEM containing 0.5% dialyzed FCS. Analysis started 15 min after addition of the energy depletion medium.
For depletion of cellular ATP (and GTP; Schwoebel et al., 2002 (link)), cells were treated with 6 mM deoxyglucose (Sigma-Aldrich) and 10 mM NaN3 in glucose-free DMEM containing 0.5% dialyzed FCS. Analysis started 15 min after addition of the energy depletion medium.
3
Glucose Uptake Measurement in Macrophages
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For glucose uptake, bone marrow–derived macrophages were incubated for 10 min at 37 °C with 1 µCi/ml 2 deoxy-D-(1-3H) glucose (Amersham) and 10 µM deoxyglucose (Sigma) in uptake buffer (140 mM NaCl, 20 mM HEPES, 5 mM KCl, 2.5 mM MgSO4*7H2O, 1 mM CaCl2*2H2O, pH 7.4). Cells were washed 3 times with ice cold PBS and lysed with 500 µl NaOH (0.2 M). Radioactivity was assessed in 300 µl cell lysate with a β-counter (Perkin Elmer) and normalized to the protein content. For glucose treatment, cells differentiated in medium with 4.5 mg/ml glucose were incubated overnight in medium with 2.5 mg/ml glucose and thereafter in medium containing 6 mg/ml glucose29 (link). Control cells received 6 mg/ml mannose instead.
4
Epimastigote Growth Dynamics under Stress
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Epimastigotes (2.5 × 106 parasites/mL) were grown at 28 °C under normoxic (physiological culture conditions) or hypoxic conditions in BHI with 10% FBS in the absence or presence of 1 mM urate (Sigma-Aldrich (St. Louis, MO, USA), 2 µg/mL oligomycin (Sigma-Aldrich, St. Louis, MO, USA), or 5.5 mM deoxyglucose (Sigma-Aldrich, St. Louis, MO, USA), for five days. Afterwards, the parasite proliferation was monitored by cell counting in a Neubauer chamber.
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