Laser injury assay was performed as described in Vallecillo-Zúniga et al. [31 (link)]. A/J−/− non-treated or treated myotubes were prepared for laser injury in 35 mm glass-bottom microwell dishes (Cat. No. P35GCol-1.0-14-C, MatTek, Ashland, MA). After washing with PBS, the myotubes were incubated in PBS enriched with 2.5 μM FM™ 1–43 dye (N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) Pyridinium Dibromide)3,5 (Thermo Scientific, Cat. No. T35356, Waltham, MA, USA) for 5 min before injury. Digested rHsGal-1 or PBS-treated Bla/J myofibers were placed onto 35 mm glass-bottom microwell dishes (Cat. No. P35GCol-1.0-14-C, MatTek) containing 300 µL of PBS. Myofibers were incubated with 2.5 μM FM™ 1–43 dye (N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) Pyridinium Dibromide)3,5 (Thermo Scientific, Cat. No. T35356, Waltham, MA, USA) for 5 min before injury. The kinetics of repair were examined by determining the total change in fluorescence intensity of FM™ 1–43 dye (ΔF/F0, where F0 is the original value at time 0) at the site of the wound for each time point relative to the pre-injury fluorescent intensity that was measured using ImageJ [31 (link)].
Fm1 43 dye
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan
FM1-43 dye is a fluorescent probe used for labeling cell membranes and monitoring membrane dynamics. It is a styryl dye that exhibits enhanced fluorescence upon binding to lipid bilayers. The core function of FM1-43 dye is to provide a way to visualize and study membrane-related processes in live cells.
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Lab products found in correlation
Ix81 microscope, by Olympus (6 mentions)
Microscopy stage top zilcs incubator, by Tokai Hit (5 mentions)
Lsm5 confocal microscope, by Zeiss (3 mentions)
Live cell imaging solution, by Thermo Fisher Scientific (3 mentions)
Slowfade gold antifade reagent with dapi, by Thermo Fisher Scientific (2 mentions)
Collagenase type 1, by Merck Group (2 mentions)
Fluoview 300 confocal microscope system, by Leica (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Celltirf, by Olympus (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Advasep 7, by Merck Group (2 mentions)
Dmi6000, by Leica (2 mentions)
Alexa fluor 647 conjugated α bungarotoxin, by Thermo Fisher Scientific (2 mentions)
Axio imager z1 microscope, by Zeiss (2 mentions)
Bfa powder, by Merck Group (2 mentions)
Lsm 880, by Zeiss (2 mentions)
A1rsi, by Nikon (1 mentions)
3d analysis module package, by Leica (1 mentions)
Lsm 5 exciter, by Zeiss (1 mentions)
45 protocols using fm1 43 dye
1
Laser-Induced Membrane Repair Kinetics
2
Laser-Induced Muscle Fiber Membrane Damage
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Flexor digitorum brevis (FDB) muscle fibers were isolated from male age-matched mice of each genotype as previously described and plated onto 35 mm glass-bottomed MatTek dishes (MatTek Corp., Ashland MA, P35G-0-10-C) in isotonic Tyrode buffer containing 1.25 mM Ca2+ (Cai et al., 2009 (link)). Membrane damage was induced in the presence of 2.5 μM FM1-43 dye (Molecular Probes, Eugene OR) using a Nikon A1 confocal laser microscope through a Plan Apo 60x H2O immersion objective. To induce damage, a 5x5 pixel area of the sarcolemma on the surface of the muscle fiber was irradiated using a UV laser at full power (80 mW, 351/364) for 10 s at t = 60 s (Cai et al., 2009 (link)). Images were captured 5 min after irradiation at 5-second intervals (Cai et al., 2009 (link)). For each image, fluorescence intensity in an area of about 200 μm2 directly adjacent to the injury site was measured using ImageJ software. To allow for statistical analysis from different experiments, data are presented as fluorescence intensity relative to the value before injury (ΔF/F0).
3
Drosophila S2 Cell Membrane Labeling
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Drosophila S2 cells were plated on Concanavalin A-coated cover slips, and treated with 5 μg/ml FM1-43 dye (Molecular Probes)/ HBSS for 1 min, and incubated for 5 min at 25 °C after a quick wash, and fixed immediately with 4% (vol/vol) PFA/HBSS without washing. Fixation was 10 min at RT. Cells were mounted with SlowFade® Gold antifade reagent with DAPI (Invitrogen).
4
Laser-Induced Muscle Membrane Repair
5
Monitoring Synaptic Vesicle Dynamics
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Enteric neurons were incubated for 60 sec at 37°C with 5 μg/mL of FM 1-43 dye (cat: T35356, Molecular Probes) diluted in stimulation buffer composed of 31.5 mM NaCl, 90 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 25 mM HEPES, 30 mM glucose, pH 7.4. Enteric neurons were then washed for 15 min at 37°C in Ca2+-free washing buffer (119 mM NaCl, 2.5 mM KCl, 4 mM MgCl2, 25 mM HEPES, and 30 mM glucose, pH 7.4). Dynamic acquisition was performed using a scanning confocal microscope NIKON A1 RSi (Nikon, Tokyo, Japan) with an oil-immersion objective (×63, NA, 1.40). Preparations were imaged during 4 min in stimulation buffer using Galvano mode at 1 fps (excitation :48nm, emission: 530 ± 20 nm) by NIS-Element software. The fluorescence intensity of FM1-43 clusters was analyzed using the Fiji software. The destaining fraction after 4 min was calculated for each cluster using the formula: 1-(Fluo at 4min/Fluo at 0s).
6
Laser-Induced Plasma Membrane Damage in MAECs
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Plasma membrane injury in MAECs was assayed by measuring laser-wounding-induced FM-143 uptake as previously described (Bansal et al., 2003 (link)). Briefly, the cells were cultured in the record chamber and membrane damage was induced in the presence of FM-143 dye (2.4 μM; Molecular Probes) with a two-photon confocal laser-scanning microscope (LSM 510; Zeiss) coupled to a 10-W Argon/Ti: 6 sapphire laser. To induce injury, a 4 μm × 4 μm area of the membrane on the surface of the MAECs was irradiated at full power for 6.4 s at t = 20 s. Images were captured beginning 20 s before (t = 0) and for 5 min after the irradiation at 10-s intervals and the fluorescence intensity at the site of the damage was measured with the Zeiss LSM 510 imaging software. MAECs that had no membrane resealing showed dye filling at the wound site over the entire course of the experiment, whereas dye influx halted within 2 min for the membrane that resealed under the experimental conditions. FM-143 dye uptake was quantified by determining dF/Fo [= (Ft−Fo)/Fo] (Ft: the mean fluorescence intensity at time t; Fo: initial fluorescence intensity) after wounding within a 40 μm × 40 μm ROI. Metafluor imaging and analysis software was used to acquire, digitize and store the images for off-line processing and statistical analysis.
7
Laser-Induced Muscle Membrane Repair
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After mice were sacrificed by cervical dislocation, FDB muscles were surgically removed to be digested in a Tyrode's solution containing type I collagenase (2 mg/ml; Sigma) at 37 °C for 60 min. After re-suspension in the Tyrode's solution, single FDB fibers were mounted on a glass bottom chamber in Tyrode's or zero Ca2+ solution in the presence of 2.5 µM green-colored FM1–43 dye (Molecular Probes). To induce damage to the muscle fibers, a selected region (5 µm × 5 µm) of the plasma membrane was irradiated for 5 s with a two-photon laser (laser power 3700 at wavelength 820 nm) in a Leica Fluoview 300 confocal microscope system. Images were captured at 10-s intervals. For every image taken, the fluorescence intensity at the site of the damage was measured with ImageJ software. Fibers that had defective membrane resealing showed dye accumulation at the injury sites throughout the time course of the experiment, whereas for resealed fibers, dye influx stopped typically within 1 min.
8
FM1-43 Dye Loading at Larval NMJs
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Third-instar larvae were dissected in Ca2+-free HL3 saline on Sylgard-coated plates. Motor nerves were stimulated at × 2 threshold using an Axoclamp 900A amplifier to load FM1-43 dye (4 μM; Molecular Probes) in HL3 solution supplemented with 1 mM Ca2+. FM dye loading of NMJs at muscle 12/13 in segments A2-A4 was imaged using a × 60 1.0N.A. water immersion lens on a Nikon fluorescent microscope. The microscope filter was set for FM1-43 emission and excitation.
9
Drosophila S2 Cell Membrane Labeling
10
Isolating and Quantifying Outer Membrane Vesicles
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Outer membrane vesicles (OMV) were isolated using a previously reported protocol22 (link). Quantification of vesicles was performed with the help of Dr. Matt Mellema, UC Davis using a Nanosight LM10HS in which a true medium blank and FM 1–43 dye from Molecular Probes were used to distinguish OMV from background particles. FM dyes are lipophilic styryl compounds used in previous studies of vesicles and plasma membranes. Note: We suggest rinsing all ultracentrifuge and storage tubes with 0.1 μm filtered ddH20 to reduce the particle background.
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