1×106 cells were plated in glucose-free RPMI 1640 media supplemented with 10% dialyzed FBS. Cells were incubated for 30 minutes to allow equilibration with media. Following incubation, cells were given 25μl of 14C deoxy-glucose (0.1μCi/μl) for one hour, then washed three times with ice cold PBS. Cells were lysed in 500 μl 0.1% SDS. 400μl of lysate was added to 5ml Microscint 40 scintillation fluid (Perkin Elmer) and counts were measured on a Tri-Carb 2910 liquid scintillation analyzer (Perkin Elmer). Remaining lysate was quantitated with the BCA Assay as described above. Counts were normalized to μg protein concentration.
Microscint 40 scintillation fluid
Manufactured by PerkinElmer
Sourced in United States
Microscint 40 is a scintillation fluid designed for use in liquid scintillation counting. It is formulated to provide high counting efficiency and low background for a wide range of sample types.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using microscint 40 scintillation fluid
1
Glucose Uptake Quantification Assay
2
Measuring Glucose Uptake in HeLa Cells
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HeLa cells were transfected with PFKFB3 siRNA as described above. Forty-eight hours later, cells were washed twice with PBS and incubated in glucose-free media for 30 min. Cells were then treated with 25 μl of 14C-deoxy-glucose (0.1 μCi/μl) for 1 h, washed once with ice-cold glucose-free DMEM and twice with ice-cold PBS. Cells were lysed in 500 μl 0.5% SDS. Four hundred microliters of lysate was added to 5 ml Microscint 40 scintillation fluid (Perkin-Elmer, Akron, OH, USA) and counts were measured on a Tri-Carb 2910 liquid scintillation analyzer (Perkin-Elmer). Remaining lysate was quantitated with the BCA Assay (Pierce) per manufacturer's instructions. Counts were normalized to protein concentration.
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