Betaplate scint

ARPE-19 cells (5×10³) were seeded in 96-well flat plate with the presence of absence of rIL-22 (10 ng/ml). Result was the representative of three independent experiments and each experiment was performed in triplicate. After 30 hr, 1 μCi of [³H]-Thymidine (American Radiolabeled Chemicals, Inc., St. Louis, MO, USA) was added to each well. After 18 hr incubation, cells were harvested onto glass fiber filters using a cell harvester (Inotech biosystems international, Dietikon, Switzerland). When dry, these were sealed into polyethylene bags with scintillation fluid (BetaplateScint; PerkinElmer, Boston, MA, USA) and incorporated [³H]-thymidine counted on a MicroBeta Trilux 1450 (PerkinElmer).

The separation of gibberellin by gradient RP-HPLC was adapted from the isocratic system described by Bhalla and Singh^{48 (link)}, where the solvent was initially 90% acetonitrile (ACN) and 10% H₂O at a flow of 0.5 mL/min for 45 min at 30 °C over a YMC C18 ODS-A column (5 µm, 300 Å, 3.0 mm × 150 mm). The wavelength used for analysis was 206 nm, and commercial gibberellin 3 (GA3) and 4 (GA4) (Sigma-Aldrich, Darmstadt, Germany) was used as a standard.
PfexG was co-injected with standard GA4, and the components were chromatographically separated by RP-HPLC. The samples were dried at 50 °C, resuspended in 0.5 mL of scintillation liquid (BetaplateScint, Perkin Elmer, Groningen, Netherlands) and analyzed in a Beckman^® scintillator unit.

L-glutamine uptake was determined by measuring the incorporation of radio-labeled L-glutamine into biotinylated-SLC1A5 proteoliposomes. Specifically, the assay was initiated by the addition of biotinylated-SLC1A5 proteoliposomes into triplicate wells of a 96-well polypropylene plate with each well containing the indicated concentration of NaCl and 50 µM L-glutamine/L-[3,4-³H)]-glutamine (100 dpm/pmol). After the indicated time at room temperature (~22°C), the assay was stopped by the addition of 15 µl of ice-cold streptavidin beads (Pierce Streptavidin Agarose) prepared in 20 mM Tris, pH 7.0, 15 mM sucrose buffer. After 10 min at room temperature the biotin proteoliposome/streptavidin bead complex was transferred to a 96-well GF/C filter plate (PerkinElmer) plate (pre-blocked with 0.1% BSA and washed x 2 in 20 mM Tris, pH 7.0, 15 mM sucrose buffer). The plate was then vacuum filtered and then washed three times with 500 µl per well of ice-cold 20 mM Tris, pH 7.0, 15 mM sucrose buffer. The plate was then air dried overnight and 50 ul of Betaplate Scint (PerkinElmer) was added to each well of the 96-well plate. The plate was then sealed and glutamine uptake was determined by scintillation counting with a MicroBeta2 (PerkinElmer).