Gf c glass fiber filter

In vitro binding assays were conducted on human opioid receptors stably transfected into CHO cells according to the published procedures [51 (link)]. Assays were performed in 50 mM Tris-HCl buffer (pH 7.4) in a final volume of 1 mL. Cell membranes (20 µg) were incubated with various concentrations of test compounds of [³H]U69,593 (0.4 nM) or [³H]diprenorphine (0.2 nM) for labeling KOR or DOR, respectively, for 60 min at 25 °C. Non-specific binding was determined using 10 µM U69,593 or 1 µM diprenorphine. After incubation, reactions were terminated by rapid filtration through Whatman GF/C glass fiber filters. Filters were washed three times with 5 mL of ice-cold 50 mM Tris-HCl buffer (pH 7.4) using a Brandel M24R cell harvester (Brandel, Gaithersburg, MD, USA). Radioactivity retained on the filters was counted by liquid scintillation counting using a Beckman Coulter LS6500 (Beckman Coulter Inc., Fullerton, CA, USA). Inhibition constant (K_i, nM) values were determined by the method of Cheng and Prusoff [66 (link)] from concentration–response curves by nonlinear regression analysis using the GraphPad Prism 5.0 Software (GraphPad Prism Software Inc., San Diego, CA, USA). All experiments were performed in duplicate and repeated at least three times with independently prepared samples.

The [³⁵S]GTPγS binding assays were performed following the procedure described elsewhere for mice and human brain membranes (Saumell-Esnaola et al., 2021 (link)). Briefly, brain cortical and hippocampal membranes were thawed, and incubated at 30°C for 2 h in [³⁵S]GTPγS-incubation buffer (0.5 nM [³⁵S]GTPγS, 1 mM EGTA, 3 mM MgCl₂, 100 mM NaCl, 0,2 mM DTT, 50 μM GDP, and 50 mM Tris-HCl, pH 7.4). The cannabinoid CB₁ receptor agonist WIN 55,212-2 (10^–9–10^–5 M, eight concentrations) was added to determine receptor-stimulated [³⁵S]GTPγS binding. Non-specific binding was defined in the presence of 10 μM unlabeled GTPγS. Basal binding was assumed to be the specific [³⁵S]GTPγS binding in the absence of agonist. The reactions were terminated by rapid vacuum and filtration through Whatman GF/C glass fiber filters and the remaining bound radioactivity was measured by liquid scintillation spectrometry as described above.

YAC-1 cells were acquired from ATCC (Catalog number TIB-160). Cells were maintained in supplemented medium as described for EL4 cells. Specific cytotoxic activity against tumor cells was determined according to the just another method (JAM method) as previously reported (7 (link)). Briefly, YAC-1 cells were cultured in the presence of 5 mCi [³H]-thymidine for 16 h. Cell suspensions from spleens of mice from different groups were obtained. Briefly, spleens were removed and disrupted through a 1-mm metal mesh, and the cell suspensions were filtered through a 10-lm nylon mesh. The suspensions were depleted of red blood and dead cells using a lysis buffer (NH₄Cl 8.29 g, KHCO₃ 1 g, EDTA-2Na 37.2 mg, diluted in distilled water, at pH = 7.4) for 2 min. After three washes in PBS, cells were re-suspended in PBS at final concentration. Cell viability was assessed by trypan blue exclusion assay. A target:effector ratio 1:50 was seeded in 96-well plates at a final volume of 200 µl, and incubated for 3.5 h at 37°C in a 5% CO₂ atmosphere. [³H]-Thymidine incorporation was measured by scintillation counting after retention over GF/C glass-fiber filters (Whatman). NK activity was calculated as 100 × (SR − ER)/SR, where SR is the spontaneous release and ER is the experimental release.

Dry weight (DW) of microalgal biomass was determined on the basis of the differences in dry weight before and after filtering the algal suspension. The microalgae werecollected on Whatman GF/C glass fiber filters, first dried at 105 ± 5 °C for 3 h, cooled in a desiccator, weighed, and used to filter 10 mL of the microalgae samples. The filters containing the microalgae suspension were again dried at 105 ± 5 °C for 3 h, cooled in a desiccator, and weighed. The density of the suspension was calculated as mg L⁻¹ DW.

Nutrients and organic matter loading was estimated by its concentrations observed in the discharge site, and the wastewater flow (18600 m³ d⁻¹) to La Salada Cove. In the discharge site, integrated water samples were collected in each season for the determination of nitrite, nitrate, ammonium, orthophosphate, biochemical oxygen demand, and chemical oxygen demand. Its concentrations were determined by chemical methods [22 ].
In La Salada Cove, water quality was sampled at 12 sampling stations each week three times in a representative month of each season: winter (February), spring (April), summer (September), and fall (November). Water samples were taken between 7:00 and 13:00 h. At each sampling station, water was collected both near the surface and near the bottom in 1 L plastic bottles; these samples were used to measure nutrients (nitrite, nitrate, ammonium, and orthophosphate) and chlorophyll a. Measurements of temperature, salinity, and dissolved oxygen were made at each station using a Hydrolab DS5X multisensor, Hach, Loveland, CO, USA. Water samples were transported on ice to the laboratory for analysis. Nutrient concentration was determined by chemical methods [22 ]. Samples for analysis of chlorophyll a were collected by filtration through Whatman GF/C glass fiber filters, extracted with 90% v/v acetone, and measured by spectrophotometry according to [23 ].