Gf c filter

The extracts were obtained from cultures grown for 25 and 70 days. Each extract was obtained from a separately grown culture. Fifty-milliliter samples were used for the preparation of cyanobacterial extracts. Culture volumes were sonicated at 4 °C using probe Ultrasonic Processor VCX 130 (Sonics, Newtown, CT., USA) until all cyanobacterial cells were disintegrated (verified under a light microscope). Homogenates were then filtered through 0.45 μm GF/C filters (Whatman, Maidstone, UK). Extracts were kept at −40 °C prior to the toxicological analyses. Simultaneously, cultures were subjected to chlorophyll-a concentration—20 mL of each culture was collected, filtered through GF/C filters (Whatman, Maidstone, UK), and analyzed spectrophotometrically following the extraction in 90% acetone [81 ]. The concentration of the extracts was standardized to be equal to 1400 μg chlorophyll per L⁻¹ to allow a direct comparison of their toxic potencies.

The cellular background HEK293 containing 400 μL of h-SERT (Code RBHSTM400UA, Perkin-Elmer, Santiago, Chile) was diluted in 12 Eppendorf tubes, containing a storage buffer solution of Tris-HCl 50 mM (pH 7.4), EDTA 0.5 mM, MgCl₂ 10 mM and 10% sucrose, obtaining a final volume between 260 and 340 μL, which was finally stored at −80 °C [46 (link)].
Each Eppendorf tube was incubated with 50 mM Tris HCl buffer (pH 7.4), 120 mM NaCl, 5 mM KCl and the drugs under study using increasing concentrations, in presence of 2 nM of [³H]-paroxetine (specific activity 23.1 Ci/mmol, Code NET86925UC, Perkin-Elmer, Santiago, Chile) with a final volume of 250 μL [46 (link)]. Non-specific binding was determined using 25 mM fluoxetine. After 30 min at 27 °C, the incubation was stopped by rapid filtration on a Whatman GF/C filter preabsorbed in 0.5% polyethylenimine (PEI), washed with cold working buffer solution, 3 × 3mL, filtered, and scintillation liquid was added. The radioactivity was measured by liquid scintillation spectrometry (MicroBeta 2450 microplate counter, PerkinElmer, Santiago, Chile). The data were represented in a bar graph using 25 μM concentration for both, the compound under study and fluoxetine as the non-specific ligand. Each bar represents the mean ± S.E.M obtained in the experiments carried out in triplicates.

The cells were grown in YPD to log phase, washed and then transferred to YNB media and incubated for additional 3 h at 30 °C. Cells were harvested, washed with cold YNB without glucose and adjusted to 10 OD/ml. Each 100 µl of cells were mixed with 100 µl of 2× YNB media with glucose supplemented with 200 µM flucytosine and 0.8 µCi of [³H] flucytosine (Moravek Inc; 4.0 Ci/mmole, 1 mCi/ml). Cells were incubated at 30° C in a water-bath shaker and 50 µl aliquots of sample were removed at 0, 1, 2, 5, 10, 20, 30, and 60 min and added to 2 mL cold media containing 20 mM flucytosine. Cells were immediately filtered with 24 mm Whatman GF/C filter and washed with cold YNB-A-N and assayed for radioactivity.