420 ac fluorescence detector

Both intracellular polyamines and polyamines generated in the culture media of the transfected cells were measured by HPLC. Transfected HEK293T cells were collected in phosphate buffered saline (PBS), pelleted, and the polyamines were extracted from the cells by treatment with 0.4M perchloric acid. The supernatant obtained after centrifugation at 10,000xg for 10 min was used for polyamine determination. For extracellular polyamine analysis, a fraction of the cell culture media was concentrated with a Speedvac Concentrator (Savant Instruments Inc. Farmingdale, NY, USA), and the resulting residue was resuspended in 0.4 M perchloric acid and processed as described above. Polyamines from the acid supernatant were dansylated according to a standard method [43 (link)]. Dansylated polyamines were separated by HPLC using a BondaPak C18 column (4.6 x 300 mm; Waters) and acetonitrile/water mixtures (running from 70:30 to 96:4 during 30 min of analysis) as mobile phase and at a flow rate of 1 ml/min. 1,6-Hexanediamine and 1,7-heptanediamine were used as internal standards. Detection of the derivatives was achieved using a Waters 420-AC fluorescence detector, with a 340 nm excitation filter and a 435 nm emission filter.

Testes were homogenized in 0.4 M perchloric acid (1:10 w/v), and the supernatants obtained after centrifugation at 12,000xg for 10 min were used for polyamine analysis by HPLC. For this purpose 100 μl of supernatant were mixed with 200 μl of saturated sodium carbonate and 400 μl of dansyl chloride (10 mg/ml in acetone) and incubated at room temperature overnight. Dansylated polyamines were extracted with toluene and separated by HPLC using a Bondapak C18 column (4.6 × 300 mm) and acetonitrile/water mixtures (running from 70:30 to 96:4 during 30 min of analysis) as mobile phase and at a flow rate of 1 ml/min. 1,6-Hexanediamine and 1,7-diaminoheptane were used as internal standards. Detection of the derivatives was achieved using a Waters 420-AC fluorescence detector, with a 340-nm excitation filter and a 435-nm emission filter.