Epi 2500 electroporator

Approximately 2.5–3.0 × 10⁶ A2lox ES cells (a gift from Michael Kyba) grown on mouse embryonic fibroblasts were harvested by trypsinization, washed with 25 ml PBS (Sigma D8537), resuspended in 125 μl PBS and mixed with 20 μg of each p2lox AE-targeting vector and Cre-expressing plasmid at 240 V, 7 ms settings on a EPI 2500 electroporator (Fischer). Immediately after electroporation, 1 ml ES cell medium with leukaemia inhibitory factor (LIF) was added to the cells, they were plated on feeder cells on a 6-cm Corning dish and were grown for 24 h without selection. ES cell medium was then supplemented with G418 (300 μg per ml) and cells were grown for 5–7 days, changing the medium every day. Individual colonies were picked and expanded on mouse embryonic fibroblasts. The clones were frozen in foetal calf serum with 10% dimethylsulphoxide and stored in liquid nitrogen.

Transfection with siRNA was performed as described [31 (link)]. In brief, cells were cultured at a low density to ensure log phase growth. For transfection, 2 × 10⁶ cells were resuspended in 200 μL RPMI 1640 without phenol red. Shortly before transfection, TRPM2 or nontargeting siRNA was added at a concentration of 1 μM. TRPM2 ON-TARGET SMARTpool and the siCONTROL NON-TARGETING pool siRNA were purchased from Dharmacon (Chicago, IL, USA). Cells were electroporated in a 4 mm cuvette in an EPI2500 electroporator (Fischer, Heidelberg, Germany) at 370 V for 10 ms. Immediately after transfection, cells were resuspended in 6 mL prewarmed medium and continued to be cultured as described above. Transfection efficiency as well as viability was determined by transfecting the cells with 400 nM green fluorescence siRNA (siGLO from Dharmacon, Chicago, IL, USA) followed by propidium iodide exclusion dye and flow cytometric analysis.