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The cells were grown in a defined medium containing DMEM/HAM F12, supplemented with 33 mM glucose, 2 mM glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, 13 mM sodium bicarbonate, 5.5 g/ml transferrin, 10 g/ml insulin, 1 pM -estradiol, 3 nM triiodothyronine, 20 nM progesterone, 5 ng/ml sodium selenite, and 100 M putrescine

A 5 L aliquot of the filtered meta -phosphoric acid extract was injected onto a Restek Ultra Aqueous C18 column (150 mm 4.6 mm, 5 m, 100 ) (Fisher Scientific Canada) attached to an Agilent 1100 HPLC coupled to a diode array detector and eluted with 20 mM ortho -phosphoric acid as described by meta -phosphoric acid extract with 0.5 volume of dithiothreitol (DTT) as described by GSH and GSSG concentrations within florets were measured spectrophotometrically with a glutathione reductase (GR)-coupled assay that reduces 5,5-dithio-bis-(2-nitrobenzoic acid) to 5-thio-2-nitrobenzoic acid ( 2 PO 4 (pH 7.5), 10 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM NADPH, 0.6 mM 5,5-dithio-bis-(2-nitrobenzoic acid), 0.2 units GR, and 20 L of the neutralized extract

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peptide reconstitution calculator tools help determine the exact volume needed to achieve specific dosing in units on an insulin syringe

Gentle swirling or rolling is preferred for delicate peptide solutions