Calcium movement and intensity within the cardiac disks were measured on the same day of cryoablation. Both calcium activities before and after the cryoablation were recorded by a DMi8 fluorescence microscope (Leica, Wetzlar, Germany). RPMI + B-27 supplement with insulin with 5 µM Rhod-2 acetoxymethyl ester (Rhod-2 AM, cat#50024, Biotium, Fremont, CA) was first used for incubating the cardiac disks at 37 °C. After 30 min, the dye solution was removed and replenished with Tyrode’s salt solution for another 30 min incubation. Imaging was performed on the microscope stage with a heating plate to ensure the temperature was at 37 °C during the process. At least three videos were recorded from each disk at randomly selected areas to generate calcium traces, which are shown in normalized intensity (F/F0) versus time, along with the average upstroke and downstroke velocities. Images were taken along the radius of each disk at regular intervals before cryoablation and across the ablated regions along the same radius after cryoablation to show the treatment effects on cardiac disks. Integrated calcium intensities were acquired from each whole image using Fiji (ImageJ; National Institutes of Health, Bethesda, MD, USA) and normalized to the highest value of each disk (F/Fmax).
Rhod 2 am
Manufactured by Biotium
Sourced in United States
Rhod-2 AM is a cell-permeant calcium indicator dye used for measuring intracellular calcium levels. It exhibits a large fluorescence increase upon binding to calcium ions.
Automatically generated - may contain errors
Lab products found in correlation
Rh237, by Biotium (2 mentions)
Blebbistatin, by Enzo Life Sciences (2 mentions)
Dmi8 fluorescence microscope, by Leica (1 mentions)
Calcium green 5n, by Thermo Fisher Scientific (1 mentions)
Entranster in vivo, by Engreen Biosystem (1 mentions)
C48 80, by Merck Group (1 mentions)
Fluo 3 am ester, by Biotium (1 mentions)
4 methylumbelliferyl n acetyl β d glucosaminide, by Merck Group (1 mentions)
H4000, by Engreen Biosystem (1 mentions)
A1r microscope, by Nikon (1 mentions)
Blebbistatin, by LGC (1 mentions)
Imidazole, by Thermo Fisher Scientific (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
Mag fluo 4, by Thermo Fisher Scientific (1 mentions)
Di 8 anepps, by Biotium (1 mentions)
Pluronic acid, by Biotium (1 mentions)
9 protocols using rhod 2 am
1
Cardiac Calcium Dynamics Quantification
2
Mast Cell Degranulation Assay
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SHL injection and its 2 intermediate fractions (ES and ELF), which were prepared according to the Chinese Pharmacopoeia23 were from Duoduo Pharmaceutical Co., Ltd. (Jiamusi, Heilongjiang, China). C48/80, 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide, and Pluonic F-127 were purchased from Sigma-Aldrich (St Louis, MO, USA). Fluo-3 AM Ester and rhod-2/AM were from Biotium (San Francisco, CA, USA). Ketotifen and cromolyn sodium were from TCI (Tokyo, Japan) and National Institutes for Food and Drug Control (Beijing, China), respectively. Calcium Green-5N and PerCP-eFluor 710 labeled anti-mouse FcεR1 antibody were obtained from Invitrogen (Carlsbad, CA, USA) and eBioscience (San Diego, CA, USA), respectively. The transfection reagents Entranster TM-in vivo and -H4000 were from Engreen Biosystem (Beijing, China). Balb/c mice (male, 18–20 g) and SD rats (male, 160–180 g) were from Vital River Experimental Animal Services (Beijing, China).
3
Calcium Handling in RyR2-E4872Q Mutant Mice
4
Cellular Signaling Detection in G. uralensis Roots
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In G. uralensis roots, we used specific fluorescent probes to detect cellular signal contents. We used Rhod-2 AM (Biotium) to detect cytosolic Ca2+ (Sun et al., 2012a (link); Zhang et al., 2015 (link)); H2DCF-DA (Eugene) to detect H2O2 (Sun et al., 2010a (link),b (link)); and DAF-FM DA (Eugene) to detect NO (Sun et al., 2012a (link)). Briefly, young roots were exposed to 0 or 100 mM NaCl for 30 min. Then, the roots were transferred to a 5 mM Mes-KCl loading buffer (pH 5.7) containing 2 μM Rhod-2 AM, 50 μM H2DCF-DA, or 10 μM DAF-FM DA. The staining was performed in the dark for 1 h at room temperature. Next, the roots were washed 4–5 times with Murashige and Skoog (MS) liquid medium prior to confocal microscope measurements. The confocal settings were as follows: excitation 488 nm, emission 510–530 nm for H2DCF-DA and DAF-FM DA; and excitation 543 nm, emission 570–590 nm for Rhod-2 AM (frame = 512 × 512).
5
Fluorescence Imaging Reagent Protocol
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Di-8-ANEPPS, Rhod-2AM, and pluronic acid were purchased from Biotium. Mag-Fluo 4 was purchased from Invitrogen. Thapsigargin (Tg) was bought from Millipore Sigma. Imidazole was from Thermo Fisher Scientific. All other drugs were from Sigma-Aldrich.
6
Detecting H2O2 and Ca2+ in Arabidopsis Roots
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Endogenous H2O2 and [Ca2+]cyt in Arabidopsis roots were specifically detected using a green fluorescent probe, H2DCF-DA (50 μM, Eugene, Shanghai, China) and an orange fluorescent probe, Rhod-2 AM (2 μM, Biotium, San Francisco, USA), respectively [6 (link),10 (link),30 (link)]. Briefly, 10 μM ACC was added to 1/2 MS liquid medium supplemented with 0 or 100 mM NaCl for 30 min. Next, the roots were transferred to a fresh MES-KCl loading buffer (5 mM, pH 5.7) containing H2DCF-DA or Rhod-2 AM, respectively, in the dark to observe the intracellular H2O2 or [Ca2+]cyt fluorescence after 1 h of incubation, when vigorous fluorescence could typically be seen [10 (link)]. After washing with 1/2 MS liquid medium 3‒4 times, the levels of endogenous H2O2 and [Ca2+]cyt in roots were visualized by confocal microscopy prior to fluorescence intensity calculation. The confocal parameters for determination of cellular signal contents were as follows: excitation at 488 nm for H2DCF-DA and 543 nm for Rhod-2 AM, emission at 510‒530 nm for H2DCF-DA and 570‒590 nm for Rhod-2 AM, and frame 512 × 512 [6 (link),10 (link),30 (link)].
7
Cardiac Electro-Calcium Imaging Protocol
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Hearts were perfused with membrane voltage-sensitive (Vm; RH237, 8-15µL of 1 mg/mL in DMSO; Biotium, Hayward, CA) and calcium-sensitive (Ca2+; Rhod2-AM, 20µL for Langendorff heart and 50 µL for innervated heart of 1 mg/mL in DMSO + 10% Pluronic acid; Biotium, Hayward, CA) indicators through coronary perfusion. The anterior epicardial surface was illuminated with LED light sources centered at 530 nm and bandpass-filtered from 511–551 nm (LEX-2, SciMedia, Costa Mesa, CA). Fluorescence emission was collected through a THT-macroscope (SciMedia, Costa Mesa, CA) and divided by a dichroic mirror at 630 nm into two paths (Omega, Brattleboro, VT). One light path longpass-filtered the RH237 signal at 700 nm and the other bandpass-filtered the Rhod2-AM signal at 574–606 nm37 (link). Individual fluorescent signals were captured using two CMOS cameras (MiCam Ultima-L, SciMedia, Costa Mesa, CA) with a field of view of 10 × 10 mm at a sampling rate of 1 kHz. The effective spatial resolution was 100 μM/pixel.
8
Optical Mapping of LV Epicardial Signals
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For optical mapping of the left ventricular (LV) epicardial fluorescence signals, Ca2+-sensitive dye Rhod-2, AM (0.2 μmol; Biotium, Inc, Fremont, CA) was slowly infused into the perfusate solution. Blebbistatin (1 μmol; Enzo Life Sciences, Inc, Farmingdale, NY) was also added to the perfusate to eliminate motion artifacts during imaging. The fluorescence was excited with a xenon light source (Moritex, Saitama, Japan) and emission light was recorded with a high-speed CMOS camera (Ultima-L; Scimedia, Costa Mesa, CA). The 1-cm2 sensor had 10,000 pixels organized in a 100 × 100 matrix, and imaging was performed at 500 frames/second using a THT splitter from Scimedia. The splitter was equipped with a Leica Planapo 0.63× lens on the objective side and a 1.0× lens on the condensing side. This provided a spatial resolution of 160 μm/pixel. Optical mapping was performed using the “pace and pause” protocol.15 (link)
9
Optical Mapping of Rabbit Hearts
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Rabbit hearts were stained with a Ca 2+ -sensitive fluorescent dye, Rhod2-AM (0.2 μM; Biotium, Inc), and a voltage-sensitive dye, RH237 (0.2 μmol/L; Biotium, Inc). To remove motion artifacts, we infused a mechanical uncoupler, blebbistatin (1 μmol/L; Enzo Life Sciences, Inc). The detailed methodology for the Langendorff setup and optical mapping has been described elsewhere. 8 (link)
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