MΦ were differentiated into M1MФ or M2MФ on μ-slide chambers (Ibidi, Biovalley). MSCs were added to polarize MΦ and labeled with Fura-2-AM (Molecular Probes). Fluorescence was quantified between 10 and 30 min on a Zeiss Axiovert 200 M inverted microscope equipped with a CCD camera (i-PentaMAX), an arc xenon lamp, and a computer-controlled monochromator (TILL Photonics) at 37°C and 5% CO2. Cells were consecutively excited with 340- and 380-nm wavelength at intervals of 10 s by means of the monochromator, and wavelength emission at 510 nm was collected with the CCD camera. The camera output was analyzed using the custom calcium-imaging software, MetaFluor, provided by Universal Imaging. Movies, and snapshots were obtained by MetaFluor and Fiji software.
Metafluor
Manufactured by Universal Imaging
Sourced in United States
Metafluor is a specialized lab equipment designed for fluorescence microscopy applications. It provides a reliable and consistent light source for fluorescence excitation, enabling researchers to visualize and analyze fluorescently labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
Fura 2 am, by Thermo Fisher Scientific (16 mentions)
Axiovert 100, by Zeiss (10 mentions)
Thapsigargin, by Thermo Fisher Scientific (9 mentions)
Metamorph software, by Universal Imaging (2 mentions)
Coolsnap hq, by Visitron (2 mentions)
Lambda dg 4, by Sutter Instruments (2 mentions)
Pluronic acid, by Thermo Fisher Scientific (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
μ slide chamber, by Ibidi (1 mentions)
Ixon ultra emccd camera, by Oxford Instruments (1 mentions)
Axiovert microscope, by Zeiss (1 mentions)
Ix70 inverted epifluorescence microscope, by Teledyne (1 mentions)
Cascade 650 digital camera, by Teledyne (1 mentions)
Calphos transfection kit, by Takara Bio (1 mentions)
Spot xplorer, by Universal Imaging (1 mentions)
Matlab, by MathWorks (1 mentions)
Metamorph, by Universal Imaging (1 mentions)
Visichrome, by Visitron (1 mentions)
Axiovert s100, by Zeiss (1 mentions)
Fluar 40x 1.3 oil, by Zeiss (1 mentions)
29 protocols using metafluor
1
Macrophage Polarization and Calcium Imaging
2
Calcium Signaling Dynamics Analysis
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Fluorescence records were analyzed using the Metafluor program (Universal Imaging). The traces shown were obtained as the ratio among the image obtained at 535nm emission and that obtained at 480nm emission. Ratio was only considered acceptable when mirror changes at both wavelengths were clearly present (Figure S1 ). Fluorescence intensities and ratio changes were then analyzed with a specific algorithm designed to calculate off-line the width at mid-height expressed in seconds, the height obtained as percent of ratio change and the frequency of all the Ca2+ peaks in each experiment. The frequency was measured at each peak as 9 divided by the distance among the peak 4 positions before and the peak 4 positions after. The mean frequency was calculated as the mean of all the individual frequencies higher than 5 peaks/min obtained in worms of a given age and condition.
3
Quantifying Synaptic Vesicle Fusion
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Video images, digitized with MetaFluor, were analyzed with MetaMorph software (Universal Imaging, USA). The fusion events of VGLUT-pHluorin positive vesicles were manually selected and counted in areas of 6000 pixels on cell surface as already reported [9 (link), 26 (link), 27 ]. A fluorescent spot was counted as “fusion event” when the pHluorin fluorescence signal of a single SLMV increased over basal by ≥4-fold.
4
Measuring Intracellular Calcium Dynamics
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Fura-2 fluorescence was utilized to determine intracellular Ca 2+ after treatment with and without istaroxime. Therefore cells were loaded with Fura-2/AM (2 µM, Invitrogen, Goettingen, Germany) for 20-30 min at 37°C and were excited alternatively at 340 nm and 380 nm through an objective (Fluor 40×/1.30 oil) built in an inverted fluorescence microscope (Axiovert 100, Zeiss, Oberkochen, Germany). Emitted fluorescence intensity was recorded at 505 nm and the data were acquired using specialized computer software (Metafluor, Universal Imaging, Downingtown, USA). Cytosolic Ca 2+ activity was estimated from the 340 nm/380 nm ratio. SOCE was determined by extracellular Ca 2+ removal and subsequent Ca 2+ re-addition in the presence of thapsigargin (1 µM, Invitrogen) [27] . For quantification of Ca 2+ entry, the slope (delta ratio/s) and peak (delta ratio) were calculated following re-addition of Ca 2+ [28, 29] .
Experiments were performed with Ringer solution containing (in mM): 125 NaCl, 5 KCl, 1.2 MgSO 4 , 2 CaCl 2 , 2 Na 2 HPO 4 , 32 HEPES, 5 glucose, pH 7.4. To reach nominally Ca 2+ -free conditions, experiments were performed using Ca 2+ -free Ringer solution containing (in mM): 125 NaCl, 5 KCl, 1.2 MgSO 4 , 2 Na 2 HPO 4 , 32 HEPES, 0.5 EGTA, 5 glucose, pH 7.4. In the DU-145 experiments, cells were treated with istaroxime (1.25µM) for 4 hours before the experiment.
Experiments were performed with Ringer solution containing (in mM): 125 NaCl, 5 KCl, 1.2 MgSO 4 , 2 CaCl 2 , 2 Na 2 HPO 4 , 32 HEPES, 5 glucose, pH 7.4. To reach nominally Ca 2+ -free conditions, experiments were performed using Ca 2+ -free Ringer solution containing (in mM): 125 NaCl, 5 KCl, 1.2 MgSO 4 , 2 Na 2 HPO 4 , 32 HEPES, 0.5 EGTA, 5 glucose, pH 7.4. In the DU-145 experiments, cells were treated with istaroxime (1.25µM) for 4 hours before the experiment.
5
Live-cell FRET Imaging of PKC Activity
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Cells were plated onto glass coverslips in 35 mm dishes, transfected with the indicated constructs, and imaged in Hanks’ balanced salt solution supplemented with 1 mM CaCl2. CFP, YFP, and FRET images were acquired with a 403 objective with a Zeiss Axiovert microscope (Carl Zeiss Microimaging) using an iXon Ultra EMCCD camera (Andor Technology) controlled by MetaFluor software version 7.10.1.161 (Universal Imaging) as described previously42 (link). For activity experiments, COS-7 cells were co-transfected with the indicated mCherry-tagged PKC construct and CKAR. Baseline images were acquired every 15 s for 2 min before ligand addition. Förster resonance energy transfer (FRET) ratios represent CFP/FRET mean ± SEM from at least three independent experiments. All data were normalized to the baseline FRET ratio of each individual cell.
6
GCaMP5-based Imaging of MSN Spine Ca2+ Dynamics
7
Calcium Imaging of Motile Sperm
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Collected sperm was loaded with 5 μM fluo-4AM, 1.5 μM Pluronic F-127 in modified sperm washing medium in the dark at 35°C for 30 min. After loading, sperm was washed once with fresh sperm washing medium. To start the experiment, sperm suspensions were added on a coverslip precoated with 10% Poly-L-lysine (0.01% w/v) for 2 min. Unattached sperm was removed by gently washing, and the chamber was filled with sperm washing medium. Treatments were added to the medium at the indicated time point as shown in the time-trace curve. Measurements were made on an Eclipse fluorescence microscope (Nikon Eclipse Ti) with a 60× oil objective lens (1.40 NA) (Nikon) and a CCD camera (Spot Xplorer) controlled by the software MetaFluor (Universal Imaging). Excitation at 488 nm was used and emission was collected at 510 nm.
8
Fura-2/AM Fluorescence for Cytosolic Ca2+ Measurement
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To determine cytosolic Ca2+ concentration ([Ca2+]i), Fura-2/AM fluorescence was utilized60 (link)–65 (link). Cells were preincubated for 15–30 minutes with Fura-2/AM (2 µM, Invitrogen, Goettingen, Germany) at 37 °C and excited alternatively at 340 nm and 380 nm in an inverted phase-contrast microscope (Axiovert 100, Zeiss, Oberkochen, Germany) through an objective (Fluor 40×/1.30 oil). At 505 nm, the emitted fluorescence intensity was recorded. Data (6/minute) were acquired using a computer software Metafluor (Universal Imaging, Downingtown, USA). To estimate cytosolic Ca2+ activity, a ratiometer (340 nm/380 nm) based analysis was employed. SOCE was determined following extracellular Ca2+ removal causing store depletion and subsequent Ca2+ re-addition in constant presence of SERCA inhibitor thapsigargin (1 µM, Invitrogen,Goettingen, Germany). For quantification of Ca2+ entry, the slope (delta ratio/s) and peak (delta ratio) were determined following re-addition of Ca2+. Experiments were performed with Ringer’s solution containing (in mM): 125 NaCl, 5 KCl, 1 CaCl2, 32 HEPES, 2 Na2HPO4, 1.2 MgSO4, 5 glucose, pH 7.4. Ca2+-free conditions were achieved by using Ca2+-free Ringer solution containing (in mM): 125 NaCl, 5 KCl, 1.2 MgSO4, 2 Na2HPO4, 32 HEPES, 0.5 EGTA, 5 glucose, pH 7.4.
9
Measuring Intracellular Calcium Levels
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Intracellular calcium levels ([Ca2+]i) were determined using a spectrofluorometric technique described previously [30 (link)]. Cultures of ciliated cells were loaded with 1.5 μM Fura-2AM (Invitrogen Corp NY, US) for 1 h at 37°C. The fluorescence of individual cells was detected at room temperature with an Olympus fluorescence microscope coupled to an image acquisition system (Metafluor, Universal Imaging Corporation, v6.1). Images were acquired at excitation wavelength of 340 and 380 nm and detected at 510 nm.
10
Analyzing Spatiotemporal Protein Dynamics
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Fluorescence resonance energy transfer images were processed using MetaMorph and MetaFluor (Universal Imaging) software and the spatiotemporal changes of fluorescent proteins in the cells were analyzed using Matlab (Mathworks, Natick, MA, USA). Firstly, all images were read and fluorescence intensity from the four corners of the image were calculated as the background. Then, background, noise, and edge recognition were removed. Images were divided into 50 parts equally along the direction of shear stress as explained in Supplementary Figure S2 . In combination to time sequence of photographing, the fluorescence intensity of each part at the previous moment of stimulation was used as a reference to normalize fluorescence intensity, after which a three-dimensional graph of time, space, and fluorescence intensity was drawn and a color bar given at the right side of the images that mentions the range of the FRET ratio [32 (link),33 (link)].
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