Human iAstrocytes were incubated with 100 nM Fingolimod (FTY720-phosphate, Selleckchem), 100 nM Siponimod (BAF312, Selleckchem) or 1 μM NIBR-0213 (Merck) or vehicle (PBS or DMSO max 0.4% v/v) for 1 hour. Cells were then treated with IL1β (10 ng/ml, Thermo Fisher Scientific), IL17 (10 ng/ml, Peprotech) or S1P (100 nM, Echelon Biosciences). Incubation times were 1 h for S1P1 internalization assay, 30 min for NFκB assay, 1, 2 or 4 h for Nrf2 assay or 24 h for glutamate transporter assay. Cells were then processed for immunofluorescence and stained with appropriate primary antibodies. For the generation of astrocyte conditioned media iAstrocytes were pre-incubated with drugs, and then exposed to the inflammatory stimuli for 8 h. Astrocyte medium was replaced with fresh neuronal medium and, after additional 24 h culture, supernatants were collected, centrifuged to remove cell debris, and stored at −80°C. Before addition to primary neurons, astrocyte supernatants were diluted down to 1:4 with medium.
Sphingosine 1 phosphate (s1p)
Manufactured by Echelon Biosciences
Sourced in United States
S1P is a laboratory product manufactured by Echelon Biosciences. It is a bioactive lipid that serves as a signaling molecule involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Il 1β, by Thermo Fisher Scientific (1 mentions)
Il 17, by Thermo Fisher Scientific (1 mentions)
Elisa kit, by Echelon Biosciences (1 mentions)
Sphingosine 1 phosphate (s1p), by Bio-Techne (1 mentions)
Fitc solution, by Merck Group (1 mentions)
Spectramax m2, by Molecular Devices (1 mentions)
Bradykinin, by Merck Group (1 mentions)
5 protocols using sphingosine 1 phosphate (s1p)
1
Pharmacological Modulation of Astrocyte Response
2
Preparation of S1P for Drug Treatment
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The manufacturer’s instructions were followed to prepare S1P, which was obtained from Echelon Biosciences Inc. (Salt Lake City, USA) under catalogue number S-2000. To summarize, S1P was dissolved in pure methanol, thoroughly mixed by vortexing, and then aliquoted in 1.5 mL tubes and kept at −20 °C. To get the needed concentration on the day of the drug treatment, the S1P was conjugated with fatty acid-free BSA (4 mg/mL) in the desired volume, incubated at 37 °C, and well mixed by a vortex.
3
Fluorescent Dye and Lipid Loading in MSVs
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In order to load the selected reporter molecule, 2 × 108 MSVs were suspended in 500 µL of fluorescein isothiocyanate (FITC) solution (Sigma–Aldrich) in distilled water (10 mg mL−1), followed by 2 h of incubation in physiological-like conditions (37°C, under mild agitation). Centrifugation at 4500 r/min for 10 min allowed for particle isolation. The collected supernatant was used to estimate the amount of absorbed reporter molecules by mass difference, using a spectrophotometer (SpectraMax M2; Molecular Devices, Sunnyvale, CA, USA), at λ = 495 nm/555 nm. The FITC-loaded particles were then lyophilized overnight and subsequently encapsulated in a 5% 50:50 PLGA shell, as described above. Following the same procedure, S1P (TOCRIS, Bristol, UK) was loaded into PLGA-MSV microspheres at a concentration of 300 µg mL−1. An enzyme-linked immunosorbent assay (ELISA) kit (ECHELON, Salt Lake City, UT, USA) was used to quantify S1P concentration in the microspheres and samples. We measured the absorbance at 450 nm and determined the S1P concentration in the collected supernatants using a standard curve.
4
Release Kinetics of S1P and VEGF from Alginate Hydrogels
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Alginate hydrogels loaded with S1P were immersed in a physiological eluent buffer consisting of EBM supplemented with 5% FBS (1 mL buffer per mL of alginate) and incubated at 37°C. Additionally, alginate hydrogels loaded with VEGF165 were immersed in a physiological eluent buffer consisting of DPBS++ (1 mL buffer per mL of alginate). Samples of buffer were collected at various time points and the tubes were replenished with fresh buffer. The amounts of S1P and VEGF released were quantified by ELISA following the manufacturers’ instructions (Echelon for S1P and R&D System for VEGF165). Samples taken from blank alginate hydrogels immersed in buffer were used to subtract background readings of S1P from the FBS content at each time point (n = 4).
5
Isolation and Characterization of Adipose-Derived Stem Cells
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ASCs were isolated from human abdominal liposuction aspirate as approved by the Cooper University Hospital Institutional Review Board Committee. Briefly, the adipose tissue from liposuction aspirate was rinsed with PBS buffer and incubated with 0.1% collagenase (Northington) for 1 hour at 37°C with vigorous shaking. After 5 minutes centrifugation (1000 × g), the top lipid layer was removed and the remaining liquid portion was centrifuged again to collect the residual cell pellet. The cell pellet was treated with 1 ml water for 10 seconds to lyse the red blood cells followed by a 3rd centrifugation with 10 ml medium. The cell pellet was then plated into T75 cell culture flask with M-199 containing 10% FBS and placed into a humidified tissue culture incubator. The next day, the floating cells were removed by medium change and the adherent ASCs were grown to confluence as passage zero cells. Passage 1 or passage 2 cells were used in this study to test the effect of S1P (Echelon), bradykinin (Sigma) and PGE (Sigma) on ASCs endothelial differentiation.
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