Tri carb 3100tr scintillation counter

HEK293 cells containing shRNAs were seeded in a 12-well plate and transfected with CXCR1 plasmids. The cells were incubated in M199 medium in the presence of 1 μCi/ml/well myo-[3H] inositol (Amersham Biosciences) for 20 h. Afterward, the medium was removed and the cells were washed with 0.5 ml Buffer A (140 mM NaCl, 20 mM HEPES, 4 mM KCl, 8 mM D-glucose, 1 mM MgCl₂, 1 mM CaCl₂, and 1 mg/ml fatty acid-free BSA). Cells were then preincubated for 30 min with Buffer A containing 10 mM LiCl followed by the addition of IL-8 at 37 °C for 30 min. The reaction was terminated with 0.5 ml of ice-cold 10 mM formic acid. After 30 min, the extracts were transferred to columns containing Dowex™ anion-exchange resin (AG-1-X8 resin, Bio-Rad). Total IPs were eluted with 1 ml of ammonium formate and 0.1 M formic acid. Radioactivity was determined using a Tri-Carb^® 3100TR scintillation counter (PerkinElmer Inc.).

Two weeks after ³³P and ¹⁵N tracer additions, plants were prepared for ¹⁴CO₂ labelling, to allow movement of carbon from plant to fungus to be quantified. A 110 µl solution of NaH¹⁴CO₃ (Perkin Elmer) containing 1.0175 MBq ¹⁴C (specific activity = 1.621 GBq/mmol) was added to a cuvette in each pot. The tops of all mesh‐windowed cores were sealed using gas‐tight rubber septa (SubaSeal) to minimize diffusion of ¹⁴CO₂ into the cores. ¹⁴CO₂ gas was liberated from the NaH¹⁴CO₃ by addition of 10% lactic acid, generating a 1.0175 MBq pulse of ¹⁴CO₂. Samples of 1 ml above‐ground gas and 1 ml below‐ground gas (via the glass wool‐filled core) were taken 1 hr after release of ¹⁴CO₂ and every 4 hr thereafter to monitor the drawdown, respiration and flux of ¹⁴C through the plant–AMF network. Gas samples were injected into gas‐evacuated scintillation vials containing 10 ml Carbosorb^® (Perkin Elmer), a carbon‐trapping compound. To this, 10 ml Permafluor scintillation cocktail (Perkin Elmer) was added, and ¹⁴C content of each sample was quantified by liquid scintillation counting (Tricarb 3100TR scintillation counter; Perkin Elmer).
Pots were maintained under cabinet conditions until detection of maximum below‐ground ¹⁴C flux (20–22 hr after ¹⁴CO₂ liberation) at which point 3 ml 2 M KOH was added to cuvettes within each microcosm to capture remaining gaseous ¹⁴CO₂.