The measurement of radiolabelled glucose uptake has been described previously (21 (link)). Briefly, glucose-free RMPI 1640 medium labelled with 0.5 µCi/ml radiolabelled 2-deoxy-d-[1-3H]glucose (Perkin Elmer) was layered over 500 µl of a 1:1 mixture of silicone oil (Sigma Aldrich) and dibutyl phthalate (Sigma Aldrich). Cells (1 x 106 per measurement) were added and allowed to take up radiolabelled 2-deoxy-d-[1-3H]glucose for 4 min. The cells were then lysed in 100 mM NaOH and the radioactivity was analyzed using a Beckman LS 6500 Multi-Purpose Scintillation Counter (Beckman Coulter). Assays were performed in duplicate or triplicate per biological replicate. For each biological replicate, data were normalized to the uptake of 2-deoxy-d-[1-3H]glucose in the IL-2-maintained CTL sample.
Protocol detail
Radiolabeled Glucose Uptake Assay
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2-deoxy-d-[1-3H]glucose
silicone oil
dibutyl phthalate
Beckman LS 6500 Multi-Purpose Scintillation Counter
Assays
Biological
Cells
Deoxy
Dibutyl phthalate
Glucose
Radioactivity
Replicate
Scintillation counter
Silicone oil
Corresponding Organization :
Other organizations : University of Dundee, Imperial College London
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Variable analysis
independent variables
- Radiolabelled 2-deoxy-d-[1-3H]glucose concentration (0.5 µCi/ml)
- Radiolabelled glucose uptake
- Glucose-free RPMI 1640 medium
- Cell number (1 x 10^6 per measurement)
- Incubation time (4 min)
- Cell lysis (100 mM NaOH)
- Radioactivity analysis (Beckman LS 6500 Multi-Purpose Scintillation Counter)
- IL-2-maintained CTL sample
- Not explicitly mentioned
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