For bulk loading of cortical neurons the calcium-sensitive dye Oregon Green Bapta-1 AM (OGB-1 AM; Molecular Probes) was first dissolved in 4 μl DMSO containing 20% Pluronic, and further diluted (1/11) in dye buffer (150 mM NaCl, 2.5 mM KCl and 10 mM HEPES (pH 7.4)) to yield a final concentration of 0.9 mM. Sulforhodamine-101 (50 μM, Molecular Probes) was added to the solution for experiments in C57Bl/6 mice to distinguish neurons and astrocytes [40 (link)]. The dye was slowly pressure injected into the right visual cortex at a depth of 150–200 μm with a micropipette (3–5 MΩ, 3–10 psi, 2–4 min) under visual control by two-photon imaging (10x water immersion objective, Olympus). Activity of cortical neurons was monitored by imaging fluorescence changes with a custom-built microscope and a mode-locked Ti:sapphire laser (Mai Tai, Spectra-Physics) at 830 nm through a 40x water immersion objective (0.8 NA, Olympus). Scanning and image acquisition were implemented in custom software (Labview, NI). The average laser power delivered to the brain was <50 mW.
Oregon green bapta 1 am
Manufactured by Thermo Fisher Scientific
Sourced in United States
Oregon Green BAPTA-1 AM is a fluorescent calcium indicator used for detecting and measuring intracellular calcium levels in living cells. It is a cell-permeable, acetoxymethyl (AM) ester form of the Oregon Green BAPTA-1 dye, which becomes fluorescent upon binding to calcium ions.
Automatically generated - may contain errors
Lab products found in correlation
Pluronic f 127, by Thermo Fisher Scientific (5 mentions)
Sulforhodamine 101, by Thermo Fisher Scientific (3 mentions)
Fluoro ruby, by Thermo Fisher Scientific (1 mentions)
Mni caged glutamate, by Bio-Techne (1 mentions)
Fluoview fv1000 confocal microscope, by Olympus (1 mentions)
Water immersion objective, by Olympus (1 mentions)
Moveable objective microscope, by Sutter Instruments (1 mentions)
Mscan, by Sutter Instruments (1 mentions)
Ab5804, by Abcam (1 mentions)
Bapta, by Thermo Fisher Scientific (1 mentions)
Fura red, by Thermo Fisher Scientific (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Anti actin, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Picospritzer 3, by Parker Hannifin (1 mentions)
Celltracker red, by Thermo Fisher Scientific (1 mentions)
Rhod 2, by Thermo Fisher Scientific (1 mentions)
Software suite, by Nikon (1 mentions)
Texas red filter cube, by Nikon (1 mentions)
Cremophor el, by Merck Group (1 mentions)
14 protocols using oregon green bapta 1 am
1
Bulk Loading of Cortical Neurons with OGB-1 AM
2
Caged Glutamate Photostimulation in Striatal Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DIV11–15 striatal cultures were loaded with Oregon green BAPTA-1 AM (Molecular Probes, Eugene, OR) as described [13 (link)]. Cells were microinjected with fluoro-ruby (3.2 mg/ml, Molecular Probes) and 20 mM 4-Methoxy-7-nitroindolinyl-caged-l -glutamate (MNI-caged-glutamate, Tocris, Avonmouth, Bristol, United Kingdom) using the single cell electroporator, Axoporator 800A (Molecular Devises, Silicon Valley, CA). Alternatively, MNI-caged glutamate was bath applied to the cells at a concentration of 200 μM. Cells were kept at 37 °C and imaged on an Olympus FluoView™ FV1000 confocal microscope with a SIM scanner. Photo-uncaging was performed using 405 nm laser with Tornado scanning within the region of interest (ROI) for 500 ms. Where indicated, the following antagonists were used at the indicated concentration: APV (100 μM); CNQX (20 μM); LY341495 (100 nM); CPCCOEt (20 μM), LY393053 (20 μM) and 2-Methyl-6-(phenylethynyl)pyridine (MPEP, 10 μM, Tocris). Calcium responses in the ROI and control areas were analyzed using MetaMorph software (Molecular Devises).
3
Visualizing Cortical Neuron Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
On the imaging day, mice were anesthetized with isoflurane and the craniotomy, marked previously, was completed for dye injection. For bulk loading of cortical neurons Oregon Green Bapta-1 AM (Molecular Probes) was first dissolved in 4 μL of freshly prepared DMSO containing 20% Pluronic F-127 (Molecular Probes) and then further diluted in 35 μL of dye buffer [150 mM NaCl, 2.5 mM KCl, and 10 mM Hepes (pH 7.4) [21 (link)]. Sulforhodamine 101 (50 μM; Molecular Probes) was added to the solution to label astrocytes [22 (link)]. The dye was slowly pressure-injected into the left visual cortex at a depth of 150–200 μm at an angle of 30° with a micropipette (4–7 MΩ, 10 psi, 8 min) under visual control by two—photon imaging (20x water immersion objective, 0.5 N.A.; Olympus). The activity of cortical cells was recorded by imaging fluorescence changes with a two-photon microscope (Moveable Objective Microscope; Sutter Instrument) and a Ti:sapphire laser (Chameleon Vision II; Coherent) at 880 nm or 1,040 nm through a 20x (0.95 N.A.; Olympus) or 25x (1.05 N.A.; Olympus) water immersion objective. Scanning and image acquisition were controlled by Sutter software (4.07 frames per second for 512 × 512 pixels, Mscan; Sutter Instrument).
4
Dendritic Cell Maturation and Calcium Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Micro-channels were coated with fibronectin (Sigma) or PLL(20)-g[3.5]-PEG(2) (SuSoS Chemical). For myosin light chain kinase inhibition, cells were incubated with different concentrations of ML7 from Calbiochem as indicated for 16 h. For Ca2+ experiments, Oregon Green BAPTA 1-AM, FuraRed, BAPTA (Invitrogen), and Thapsigargin from Calbiochem were used. For IP3R inhibition, 5 μM xestospongin C from Calbiochem was used. For dendritic cell maturation, we incubate the cells 24 h with 100 ng/ml LPS (Sigma). For flow cytometry analysis, we used a homemade 24G2 anti-Fc Receptor antibodies, rabbit serum from Agro Bio as a control, and anti-CD11c (HL3 clone), anti-IAbb (AF6-120.1 clone), and anti-CD86 (GL1 clone). For immunoblot, we used anti-IP3R type 1 (Abcam ab5804), anti-IP3R type 3 (610313 BD Transduction Laboratories), anti-phospho-myosin light chain (Rockland 600-401-416), and anti-actin (Millipore). For lentivirus production, HEK cells were transfected using GeneJuice (Novagen).
5
Calcium Imaging of Granule Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Granule neuron activity in the inner granule layer (IGL) was monitored using the calcium-sensitive indicator Oregon Green BAPTA 1- AM (OGB) (O6807, Invitrogen). OGB was prepared by diluting to 5 mM with 10% (wt/vol) pluronic (P6867, Invitrogen) in dimethyl sulfoxide (276855, Sigma). OGB was delivered to the cerebellar corpus of anesthetized fish using five to ten 100-300 msec duration pulses (Picospritzer III, Parker Hannifin) through a borosilicate microcapillary (1B100F-4, WPI) with a tip diameter of 2–4 μm. Granule cells in the separate lateral (eminentia granularis) and caudal (lobus caudalis) subgroups were not reliably labeled with these injections and will be investigated in future work. Injections and subsequent experiments were performed in 10% (vol/vol) glucose-free Evans medium (in mM): 134 NaCl, 2.9 KCl, 2.1 CaCl2, 1.2 MgCl2, and 10 HEPES, pH 7.8. After injection, zebrafish were allowed at least four hours for dye-loading and recovery from anesthesia.
6
Primary Mouse HSC Calcium Mobilization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary mouse HSCs were washed and incubated in the dark with 3 mM Oregon green BAPTA-1 AM(Thermo Fisher, Beijing, China) in the presence of 1% pluronic acid for 45 min at 37 °C. The excess of fluorochrome was removed by two washing steps. HSCs were then resuspended at densities of 104 cells per well in DMEM medium and incubated with 10 mM ENMD-1068 or with an equivalent volume of dimethylsulfoxide (DMSO) before inducing internal calcium mobilization with 0.2 U/mL trypsin, 10 μΜ SLIGRL-NH2 or 10 μΜ TFLLR-NH2 (PAR1 agonist). Changes in fluorescence were recorded as previously described12 (link).
7
Optical Recording of Neuronal Activity in Zebrafish
8
Multimodal 3D Tumor Imaging and Irradiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To test the integrated system, we used a series of 3D tumor models developed at the Center for Radiological Research [53 (link)–55 (link)]. Human U87 Glioblastoma cells were chosen as an aggressive tumor model type known to grow rapidly in the 3D model system. These cells are representative of a hard-to-treat tumor that is a prime candidate for HIRT. As described previously [56 (link)], to form 3D cell cultures, human U87 Glioblastoma cells were grown as a monolayer, trypsinized, injected into the gel matrix and incubated for 24-48 h before imaging. Depending on the cell type, these injected cell boluses can form either a solid tumor core or a more dispersed cell distribution. U87’s high motility and potential for tissue invasion form a more dispersed cell bolus.
We tested the combined imaging and irradiation system with a range of cell labelling strategies: Cyto-Red (Biosettia) and CellTracker Red (Thermo Fisher) for whole cell labeling, and Oregon Green BAPTA-1 AM (Thermo Fisher), a calcium sensitive fluorescent dye as a functional reporter cellular Ca2+ dynamics. We also tested human U87 cells transfected with the pGreenFire 2.0 NFkB reporter (SBI).
We tested the combined imaging and irradiation system with a range of cell labelling strategies: Cyto-Red (Biosettia) and CellTracker Red (Thermo Fisher) for whole cell labeling, and Oregon Green BAPTA-1 AM (Thermo Fisher), a calcium sensitive fluorescent dye as a functional reporter cellular Ca2+ dynamics. We also tested human U87 cells transfected with the pGreenFire 2.0 NFkB reporter (SBI).
9
Pseudo-nitzschia fraudulenta Calcium Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pseudo-nitzschia fraudulenta cells were harvested, placed in a µ-dish (Ibidi) and stained for 60 minutes with Oregon Green BAPTA-1 AM (Thermo Fisher) at 10 µM with a final concentration of 0.1% DMSO and observed immediately using a DeltaVision System. Autofluorescence was acquired in the red channel under blue excitation and Oregon Green was acquired in the GFP channel.
10
Calcium Imaging of Cortical Spheroids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Calcium imaging was performed as previously described (Sevetson, Theyel, and Hoffman-Kim 2021) (link). Briefly, cortical spheroids were incubated for 25 minutes in complete cortical media containing 5.2μM Oregon Green BAPTA-1 AM (ThermoFisher) and 0.02% wt/vol Pluronic F-127 (ThermoFisher). After washing once with warm complete cortical media, the spheroids were then transferred to a confocal dish. Samples were kept at 37 o C throughout the imaging process.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!