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Freeze/thaw cycles degrade peptides, so aliquot the reconstituted solution if you plan to run assays over several weeks

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Cells were seeded at a density of 5000 cells per well in 200 l fresh culture medium (DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution) into 96-well flat-bottomed microtitre plates (Costar, Corning, USA) and incubated at 37 C in humidified 5% CO 2 using AutoFlow NU-5510 Direct Heat CO 2 Incubator (NuAire, USA) for 24 hours before treatment