And 3H levels were measured on a Beckman LS6500 multi-purpose scintill…

And 3H levels were measured on a Beckman LS6500 multi-purpose scintillation counter (Beckman-Coulter, Brea, CA). Calcium-dependent release was determined by subtracting baseline radioactivity secreted from cultures in the absence of Ca2+, and expressed as a percentage of the total cellular radioactivity. Total cellular radioactivity was calculated by adding the 45Ca2+ cpm released upon stimulation with high KCl and the 45Ca2+ cpm present in the resting medium with low KCl before and after stimulation.Direct Ca2+ uptake assay (Fluo-4 DirectTM assay kit)CULTEX system that ends on a hyperboloid-shaped air distribution (trumpet) ensuring uniform exposure of the aerosol on the surface of the cell culture [23-27]. The outlet of the exposure system was passed through a decontamination solution. After exposure to air or CG, the cells were fed with fresh medium from the upper side and free intracellular Ca2+ was monitored by the Fluo-4 Direct Assay described above. To assess intracellular free Ca2+ changes due to CG exposure, undifferentiated PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/15501003 or differentiated M17 cells were first loaded with Fluo-4 and an initial fluorescence reading was taken to record the basal Ca2+ level (i.e., the resting intracellular Ca2+ levels without any treatment). The cells were then exposed to either 0 ppm (air) or 16 ppm of CG (in air) for 8 min at a flow rate of 8.13 ml/well/min. The free intracellular Ca2+ changes were monitored directly following exposure as a function of time.Treatment with Ca2+ ionophore AThe Ca2+ uptake assay kit (Fluo-4 DirectTM, Invitrogen, Carlsbad, California) contains a fluo-4 analog that produces a large fluorescence intensity increase in response to Ca2+ binding with fluorescence excitation at 495 nm and emission at 516 nm. The Fluo-4TM Direct Ca2+ Assay kit was used according to the manufacturer's instructions. Briefly, 2X Fluo-4 Direct Ca2+ assay buffer (1M HEPES in Hanks buffered saline solution, pH 7.3) was thawed and mixed with 5 mM probenecid. The assay was conducted in monolayer cultures. The 2X Fluo-4 Direct Ca2+ assay buffer with 5 mM probenecid was added in equal volume to the sample volume and incubated at 37 for approximately one hour (Fluo-4 loading of cells). The fluorescence (absolute units) was measured using the SpectraMax Gemini EM (MDS Analytical Technologies, Toronto, Canada) using excitation and emission wavelengths mentioned above.Phosgene (CG) exposureThe Ca2+ Ionophore A23187 (Invitrogen, Carlsbad, California) was prepared at stock concentration in DMSO according to manufacturer's instructions. Capivasertib Further dilutions were prepared in M17 medium. A23187 was added directly to M17 cells cultured in transwell inserts to obtained final concentration of 5 M. To assess intracellular free Ca2+, the cells were first loaded with Fluo-4 prior to addition of A23187 and the change in intracellular Ca2+ concentration was measured directly following addition of A23187 using Fluo-4 Direct Assay.ResultsMorphological changes and possible synaptic activityCULTEX air/liquid exposure system for either 12 mm or 24 mm culture filters was purchased from VetroCell (Germany). Prior to exposure to CG, the cells grown on these filters were transferred into the exposure device. Cells were nourished by culture growth medium below the filter membrane and exposed directly to either air (unexposed control samples) or air/CG (experimental samples) without growth medium on top of the cells. The vapor exposure system was moved to a chemical agent expo.