U251 cells were plated at the concentration of 1.5 × 103 cells/mL and used on the third day of culture. Before experiments, cells were incubated with FURA-2 AM (3 μM; Sigma-Aldrich, Sigma Aldrich®, St. Louis, MO, USA) for 45 min and extensively washed with a Ringer solution of the following composition (in mM): NaCl 106.5, KCl 5, CaCl2 2, MgCl2 2, MOPS 5, glucose 20, Na gluconate 30, at pH 7.25 (all from Sigma Aldrich®, St. Louis, MO, USA). Cells were continuously perfused using a gravity-drive perfusion system, with tubing connected to a final tip of 100 to 200-μm diameter focally oriented onto the field of interest. UiO-66_N were added at the concentration of 1 μg/mL after 9 min of perfusion for 8 min. A standard control pressure perfused the cells for about 20 min with only the Ringer solution. A positive control pressure perfused a solution of ionomycin calcium salt (Tocris Bioscience, Bristol, UK) for about 7 min. The estimation of intracellular free Ca2+ concentration was reported as change of the ratio between fluorescence emission at 510 nm, obtained with 340 and 380-nm excitation wavelengths (optical filters and dichroic beam splitter were from Lambda DG4, Shutter Instruments, Novato, CA, USA). Ratiometric data was randomly acquired from 30 cells every 3 s.
Ionomycin calcium salt
Manufactured by Bio-Techne
Sourced in United Kingdom
Ionomycin calcium salt is a laboratory reagent used in cell biology research. It is a calcium ionophore that increases intracellular calcium levels, which can be used to study calcium-dependent cellular processes.
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Lab products found in correlation
Forskolin, by Bio-Techne (2 mentions)
Fura 2 am, by Merck Group (1 mentions)
Mgcl2 2, by Merck Group (1 mentions)
Mycoplasma testing, by Lonza (1 mentions)
Renca, by American Type Culture Collection (1 mentions)
Thp 1, by American Type Culture Collection (1 mentions)
Csf1r, by Cell Signaling Technology (1 mentions)
H1299, by American Type Culture Collection (1 mentions)
Anti pd 1 antibody, by BioXCell (1 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
Phospho erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Ionomycin, by Bio-Techne (1 mentions)
Ym 58483, by Bio-Techne (1 mentions)
Thapsigargin, by Adipogen (1 mentions)
Quinpirole hydrochloride, by Bio-Techne (1 mentions)
Snc80, by Bio-Techne (1 mentions)
Skf81297, by Bio-Techne (1 mentions)
5 protocols using ionomycin calcium salt
1
Intracellular Calcium Dynamics in U251 Cells
2
Analysis of Receptor Signaling Pathways
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Cell lines were purchased as follows: H1299, A549, THP-1, M-NSF-60, EMT6, CT26, and RENCA (ATCC, Manassas, VA, USA), B16F10-OVA (Crownbio, San Diego, CA, USA), MC38 (BioVector NTCC Inc., Beijing, China), and all cells were confirmed to be pathogen-free (including Mycoplasma testing; Lonza; #L108-318). Q702 was synthesized using Qurient Co., Ltd. (Seongnam-si, Korea). Anti-PD-1 antibody was purchased from Bio X Cell (West Lebanon, NH, USA; #BE0146, clone RMP1-14). The following antibodies were used in western blotting: phospho-Axl-(Tyr702) (Cell Signaling Technology, Danvers, MA, USA; #5724, clone D12B2), Axl (Cell Signaling Technology; #8661, clone C89E7), phospho-AKT(Ser473) (Cell Signaling Technology; #4060S, clone D9E), AKT (Cell Signaling Technology; #9272S), phospho-Mer(Tyr749/Tyr753/Tyr754) (Abcam, Cambridge, UK; ab14921), Mer (Cell Signaling Technology; #4319, clone D21F11), phospho-CSF1R(Tyr723) (Cell Signaling Technology; #3155S, clone 49C10), CSF1R (Cell Signaling Technology; #3152S), phospho-ERK1/2(Thr202/Tyr204) (Cell Signaling Technology; #4370S, clone D13.14.4E), ERK1/2 (Cell Signaling Technology; #4695S, clone 137F5), and β-actin (Sigma-Aldrich, St. Louis, MO, USA; #A5441). PMA was purchased from Sigma-Aldrich (#P1585), and ionomycin calcium salt was purchased from Tocris Bioscience (Bristol, UK; #1704).
3
Ionomycin-Induced Calcium Signaling in Spinal Cord
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ionomycin calcium salt was purchased from Tocris Bioscience (Minneapolis, MN, USA) and used per the manufacturer’s recommendations. ionomycin was dissolved in DSMO first to make a 2 mM stock concentration. ionomycin was then dissolved in 2 mM Ca2+ aCSF at a final concentration of 1, 3, or 5 µM and perfused for 1 hour. Similarly, ionomycin was dissolved in zero Ca2+ aCSF for a final concentration of 3 µM and perfused for 1 hour. After 1 hour, the ionomycin buffer was washed out with either zero or normal 2 mM Ca2+ aCSF and images were collected every 15 minutes for an additional hour. To inhibit store-operated Ca2+ entry (SOCE), we pretreated spinal cords for 30 minutes with YM-58483 (an established blocker of SOCE; 500 nM; Tocris Bioscience) prior to ionomycin perfusion in aCSF. Thapsigargin, an irreversible Sarco-Endoplasmic Reticulum Ca2+ ATPase inhibitor, was purchased from AdipoGen Life Sciences (San Diego, CA, USA) and used per the manufacturer’s recommendations. Thapsigargin was dissolved in DMSO to make a 5 mM stock solution and further diluted in 2 mM Ca2+ aCSF to make a final concentration of 1 µM to pretreat the spinal cord for 60 minutes to empty calcium stores.
4
Pharmacological Agents in Cell Signaling
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DAMGO, U50488H, SNC80, CGS21680, AVP (arginine vasopressin), IBMX, Forskolin, stromal cell-derived factor 1 (SDF-1 or CXCL12), carbamoylcholine chloride (carbachol), quinpirole hydrochloride, SKF 81297, ionomycin calcium salt, were purchased from Tocris Bioscience (Bristol, UK) and diluted in DMSO (diméthylsulfoxyde) at 10−2 M (except SDF1 at 125 µM and IBMX at 200 mM) for frozen stock aliquots and coelenterazine H substrate from Interchim (Montluçon, France) was diluted in 100% ethanol and kept at −20°C. Protease/Phosphatase Inhibitor Cocktail were purchased from Cell Signaling Technology (Leiden, Netherlands). Pertussis toxin (PTX), purchased from Tocris, were diluted in water at 0.1 µg/µl and stored at 4°C. Morphine HCl was purchased from Francopia (Paris, France). Phenylmethanesulfonyl fluoride (PMSF) was diluted in isopropyl alcohol at 200 mM and stored at −20°C. Compound 19 was generously synthetized by Domain Therapeutics (Illkirch, France). For in vitro studies, it was diluted in DMSO (diméthylsulfoxyde) at 10−2 M and frozen at −20°C. For in vivo studies, Compound 19 was kept as a powder at 4°C and diluted in a saline solution before ICV administration (NaCl 9%).
5
Pharmacological Toolkit for Neuroscience
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Diltiazem-HCl (100 mM in distilled H2O [dH2O]), D-AP5 (50 mM in dH2O), lidocaine (100 mM in DMSO), (S)-(-)-Bay K 8644 (50 mM in DMSO), TTA-P2 (10 mM in DMSO), verapamil-HCl (100 mM in dH2O), ω-conotoxin GVIA (1 mM in dH2O), ω-conotoxin MVIIC (300 µM in dH2O), and ω-agatoxin IV A (200 µM in dH2O) were from Alomone Laboratories and dissolved as indicated in parentheses. AIP (500 µM in dH2O), H-89 dihydrochloride (10 mM in DMSO), and U0126 (50 mM in DMSO) were from Calbiochem. IBMX (100 mM in DMSO), 5-HT (10 mM in dH2O), fentanyl (10 mM in dH2O), NB001 (75 mM in dH2O), NKY80 (100 mM in DMSO), and oxycodone hydrochloride (10 mM in dH2O) were from Sigma-Aldrich. FK 506 (50 mM in DMSO), forskolin (10 mM in DMSO), ionomycin calcium salt (10 mM in DMSO), KN-92 (10 mM in DMSO), KN-93 (10 mM in DMSO), [Leu5]-enkephalin (1 mM in 0.1% BSA in dH2O), SQ22536 (50 mM in DMSO), and ST 034307 (50 mM in DMSO) were from Tocris. Rp-cAMPS-pAB and 4-ABnOH (10 mM in DMSO) were synthesized and provided by F. Schwede (BioLog, Bremen, Germany).
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