Auxin accumulation assays in tobacco BY-2 suspension cells

Tobacco BY-2 cells (Nicotiana tabacum L., cv. Bright Yellow-2; Nagata et al., 1992) were cultivated as described previously (Petrášek et al., 2003). Auxin efflux was measured by cellular changes in accumulation of radioactive 1-naphthaleneacetic acid (NAA) ([³H]NAA) (Petrášek and Zažímalová, 2006). The accumulation of 2 nM [³H]NAA (American Radiolabeled Chemicals, Inc.) in cells treated with AEX or ACC was determined by liquid scintillation counting (Packard Tri-Carb 2900TR scintillation counter; PerkinElmer). Cell surface radioactivity was corrected by subtracting counts of aliquots collected immediately after addition of [³H]NAA. Counts were converted to pmol of [³H]NAA per 1 million cells.