For all calcium imaging experiments, Fluo-4AM (Invitrogen) was mixed with 10 μl Powerload (Invitrogen) and vortexed for 15 s. Fluo-4AM/Powerload mix was subsequently mixed with aCSF for a final concentration of 10 μM Fluo-4AM. Individual coverslips were removed from culture media and incubated in 600 μl of Fluo-4AM mixture for 1 h at 37 °C. Following incubation, coverslips were placed in a recording chamber on an Olympus BX51W microscope and rinsed for 15 min with continuous flow of aCSF before imaging. Cells were imaged at 470 nM (Cairn OptoLED). Areas of the coverslip with >10 healthy looking cells with neuronal morphology were chosen for imaging. Calcium signals were acquired using Turbo-SM software and an SM-CCD67 camera (RedShirtImaging, Decatur, GA, USA). For spontaneous activity, images were acquired for 6 min at 100 Hz. For plasticity experiments, images were acquired for 90 min (see plasticity protocol above) at 10 Hz. Following data acquisition, all experiments were randomized and coded and then analysed by a blinded experimenter. Cells with neuronal morphologies were chosen as regions of interest across all experiments. Traces were then blindly analysed offline in Clampfit (Axon Instruments). For 100 Hz experiments, transients were counted if they had rise times ≤15 s. For 10 Hz experiments, transients were counted if they had rise times ≤120 s.
Powerload
Manufactured by Thermo Fisher Scientific
Sourced in United States
PowerLoad is a centrifuge loading device designed to facilitate the loading and unloading of centrifuge rotors. It provides a stable platform for secure handling of rotors, reducing the physical effort required during the process.
Automatically generated - may contain errors
Lab products found in correlation
Probenecid, by Thermo Fisher Scientific (4 mentions)
Ionomycin, by Merck Group (3 mentions)
Fluo 4 am, by Thermo Fisher Scientific (3 mentions)
Fluovolt, by Thermo Fisher Scientific (3 mentions)
Indo 1 am, by Thermo Fisher Scientific (2 mentions)
F ab 2 rabbit anti mouse igg crosslinker, by Jackson ImmunoResearch (2 mentions)
Facsaria 2, by BD (2 mentions)
Fixable live dead, by Thermo Fisher Scientific (2 mentions)
Pzap70y319, by BD (2 mentions)
Other phospho antibodies, by Cell Signaling Technology (2 mentions)
Disbac43, by Thermo Fisher Scientific (2 mentions)
Fortessa flow cytometer, by BD (2 mentions)
F ab 2 rabbit anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Facsaria, by BD (1 mentions)
Pbfi am, by Abcam (1 mentions)
Pbfi am, by Thermo Fisher Scientific (1 mentions)
Pluronic f 127, by Thermo Fisher Scientific (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Bx51w microscope, by Olympus (1 mentions)
Clampfit, by Molecular Devices (1 mentions)
17 protocols using powerload
1
Calcium Imaging Experiment Protocol
2
Calcium Flux in T Lymphocytes
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Calcium influx into total T lymphocytes from freshly thawed PBMCs was analyzed by real-time flow cytometry, as previously described (Hauck et al., 2012 (link)). Briefly, cells were loaded with indo-1 acetoxymethyl ester (Molecular Probes, 5 μM) in the presence of 2.5 mM probenecid (PowerLoad; Molecular Probes), washed, surface-stained for CD4 and CD8, washed, stimulated with anti-CD3 mAb (Clone: OKT3, 1 μg/ml) followed by F(ab′)2 rabbit–anti-mouse IgG (Jackson Immunoresearch, 10 μg/ml), and then further stimulated with ionomycin (Sigma-Aldrich, 1 μM). Cells were analyzed with a FACSAria flow cytometer (BD Biosciences). Intracellular Ca2+ levels were quantified by calculating the normalized ratio of fluorescence intensity (Ca2+-bound to Ca2+-free Indo-1) and plotted as a function of time.
3
Quantifying Intracellular Calcium Dynamics
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Ca2+ influx was assessed by real-time flow cytometry, as previously described (Martin et al., 2014 (link)). Briefly, cells were loaded with 5 mM Indo-1 AM (Molecular Probes) in the presence of 2.5 mM probenecid (PowerLoad; Molecular Probes), washed, and surface-stained for CD4 and CD8 detection. Cells were analyzed in real-time with a FACS ARIA II flow cytometer (BD Biosciences). During acquisition, 1 mg/ml of anti-CD3 antibody was added to the cells, followed by 10 μg/ml of F(ab′)2 rabbit–anti-mouse IgG crosslinker (Jackson Immunoresearch), and finally incubated with a calcium ionophore (1 mM ionomycin [Sigma-Aldrich]). Changes in the intracellular calcium concentration are quantified by a shift in the indo-1 emission peak from 485 nm (indo-blue) for unbound dye to 405 nm (indo-violet) when the indo-1 molecule is bound to calcium. Data were analyzed by using the kinetic tool of FlowJo software. Intracellular Ca2+ levels correspond to the normalized ratio of 405- to 485-nm indo-1 emission peaks.
4
Real-time Flow Cytometric Analysis of Ca2+ Influx
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Ca2+ influx was assessed by real-time flow cytometry, as previously described (Martin et al., 2014 (link)). Briefly, cells were loaded with 5 µM Indo-1 AM (Molecular Probes) in presence of 2.5 mM of probenecid (PowerLoad; Molecular Probes), washed, and surface stained for CD4 and CD8 detection. Cells were analyzed in real time with a FACS ARIA II flow cytometer (BD Biosciences). During acquisition, 1 µg ml−1 anti-CD3 antibody was added to the cells, followed by 10 µg ml−1 of F(ab′)2 rabbit–anti-mouse IgG cross-linker (Jackson ImmunoResearch) and finally incubated with a calcium ionophore (1 mM ionomycin; Sigma). Changes in the intracellular calcium concentration are quantified by a shift in the indo-1 emission peak from 485 nm (indo-blue) for unbound dye to 405 nm (indo-violet) when the indo-1 molecule is bound to calcium. Data were analyzed using kinetic tool of FlowJo software (TreeStar). Intracellular Ca2+ levels correspond to the normalized ratio of 405 nm/485 nm indo-1 emission peaks.
5
Intracellular Potassium Efflux Monitoring
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Potassium efflux was confirmed by investigating the intracellular K+ content of the cells using the cell permeable, fluorophore PBFI-AM (Abcam, Cambridge, United Kingdom). After 48 h infection or treatment with the porphyrin extract, supernatant was removed and the cells were washed with Hanks balanced salt solution (HBSS; Gibco). 400 μL loading buffer (PowerLoad, Invitrogen) was added to the wells and 10 μM PBFI-AM and 10 μM Pluronic F-127 (Invitrogen) were included per well, after which the plate was incubated, protected from light, for 2h at 37°C. Afterwards, the loading buffer was removed and the cells were washed with HBSS. Imaging was accomplished using the EVOS FL Auto Imaging System equipped with a 10x objective (excitation: 357 nm, emission: 447 nm, final magnification: 300x). Fluorescence intensity was measured using the ImageJ software.
6
Multiparametric Analysis of T Cell Activation
7
Multiparametric Analysis of T Cell Activation
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Suspensions containing T cells were stained with fixable live/dead (Invitrogen) in PBS followed by surface antibody staining in FACS buffer (PBS with 0.5% BSA and sodium azide). For intracellular cytokine staining, cells were stained for intracellular molecules following fixation and permeabilization. For phosphostaining BD PhosFlow reagents were utilized and fixation/permeabilization protocols were carried according the to manufacturer’s protocol. After washing, cells were stained with antibody-fluorochrome conjugates for the indicated phosphorylated proteins (pZap70Y319 (BD Biosciences), all other phospho-antibodies were purchased from Cell Signaling). Antibodies for surface staining and intracellular cytokine staining were purchased from BD Biosciences and eBiosciences. For determination of cytoplasmic membrane potential (Vm) cells were incubated in 2uM DiSBAC43 (Invitrogen) in conditions as indicated for 60 minutes prior to evaluation. For determination of [K+]i, cells were loaded with the potassium sensitive dye Asante Green-4 (TEFLabs) with PowerLoad (Invitrogen) per the manufacturer’s protocols. All experiments were conducted on a BD Fortessa flow cytometer (Becton Dickinson) and analyzed with FlowJo software.
8
Intracellular Calcium Dynamics in αTC1.6 Cells
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αTC1.6 cells were seeded into a 96-well plate (Greiner, Frickenhausen, Germany) at a density of 2.0 × 104 cells per well and were then preincubated in KRB containing 20 mM glucose, 5 μM fluo-4/AM, 5 mM probenecid, and 1 × Power load (all from Invitrogen) for 30 min at 37 °C. Preincubated KRB was replaced with KRB containing 20 mM glucose ± 1 μM immepip and the 96-well plate was placed into a microplate reader FlexStation 3 (Molecular Devices, Sunnyvale, CA, USA) to record changes in fluorescence (excitation: 495 nm/emission: 518 nm). Cells were excited at 495 nm and the light emitted through a 518-nm filter was detected. After a 1-min measurement of baseline fluorescence, glucose concentration in KRB in each well was diluted to 2.8 mM by adding KRB ± 1 μM immepip from injectors, or KRB containing high KCl ± 1 μM immepip was added from injectors to adjust KCl concentration to 20 mM. Fluorescent intensity before fluo-4/AM loading was measured as background.
9
Voltage-sensitive Dye Imaging of Microtissues
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Voltage experiments were conducted on day 11. The microtissues were washed in serum-free medium consisting of DMEM (Gibco, Paislay, UK) supplemented with 10 mM D-Galactose (Sigma) and 1 mM sodium pyruvate (Sigma). The microtissues were loaded with voltage-sensitive dye: 0.1% FluoVolt and 1% PowerLoad (ThermoFisher, Waltham, WA, USA) in the above listed serum-free medium for 25 min at 37 °C 5% CO2. The voltage-sensitive dye was removed by washing in serum-free medium, and the multiwell plate was placed in an environmentally controlled stage incubator (37 °C, 5% CO2, >75% humidity) in the CellOPTIQ® platform (Clyde Biosciences, BioCity Scotland) 30 min before experimentation. The fluorescent signal was recorded with an excitation at 470 nm, and emitted light was collected from the entire microtissue using a 10× Fluor objective and the intensity recorded by a photomultiplier tube at 510–560 nm at 10 kHz. A 15 s recording was taken of each microtissue. Offline analysis was performed using CellOPTIQ®.
10
Cytosolic Calcium Monitoring in Human Macrophages
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