Live cell imaging and calcium mobilization experiments were performed as described previously [1 (link),19 (link)]. Cells were preincubated with ARL 67156 trisodium salt (ARL) (Sigma-Aldrich (St. Louis, MO, USA)) (10, 20, 50 nM) [25 (link),26 (link)], Fluo-4AM (Thermo Fisher, Waltham, MA, USA) (1:100) and the counterstain SirActin (Cytoskeleton, Inc., Denver, CO, USA) (1:1000) for 20 min at 37 °C. Images were collected every 3 s for 45 min on the Zeiss Axiovert LSM 880 confocal microscope (Zeiss, Thornwood, NY, USA) utilizing the 20× air objective. Scratch wounds or agonist stimulation by ATP, 2,3-O-(4-benzoylbenzoyl)-ATP (BzATP) (Sigma-Aldrich (St. Louis, MO, USA)), or ATP (Sigma-Aldrich (St. Louis, MO, USA)) were performed after 100 frames (5 min). All injuries were made using a 25-gauge needle.
Axiovert lsm 880 confocal microscope
Manufactured by Zeiss
Sourced in United States
The Zeiss Axiovert LSM 880 is a confocal microscope designed for high-resolution imaging. It utilizes a laser scanning system to capture detailed images of samples with improved contrast and resolution compared to traditional optical microscopes.
Automatically generated - may contain errors
Lab products found in correlation
Sir actin, by Cytoskeleton (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
2 3 o 4 benzoylbenzoyl atp bzatp, by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Imagej, by MathWorks (1 mentions)
Axiovert lsm 880, by Zeiss (1 mentions)
Matlab, by MathWorks (1 mentions)
Cellmask deep red, by Thermo Fisher Scientific (1 mentions)
A438079, by Bio-Techne (1 mentions)
10panx, by Bio-Techne (1 mentions)
Ar c 118925xx, by Bio-Techne (1 mentions)
4 protocols using axiovert lsm 880 confocal microscope
1
Live Cell Calcium Imaging of ATP Signaling
2
Visualizing Cell Migration and Cytoskeleton Dynamics
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To examine cell migration and alterations in cell shape, HCLE cells were pre-loaded with either CellMask Deep Red Plasma membrane stain as described above or 1 μM of SiR-Actin Spirochrome probe (Cytoskeleton Inc., Denver, CO) for 10 minutes at 37°C and 5% CO2, for imaging of F-actin. Both long- and short-term studies were performed. For long-term studies, images were collected immediately after injury and every 5 minutes for 6 hours on a Zeiss Axiovert LSM 880 confocal microscope. For short-term studies, images were taken immediately after injury and every 5 seconds for up to 2 hours on a Zeiss Axiovert LSM 880 confocal microscope. Analyses for all the described image studies were performed using FIJI/imagej (NIH, Bethesda, MD; imagej.nih.gov/ij/">http://imagej.nih.gov/ij/ ) along with MATLAB programs (MATLAB, MathWorks, Inc.) written for the analysis described below.
3
Calcium Mobilization in Corneal Cells
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Ca2+ mobilization experiments were performed on ex vivo mouse corneas [18 (link)]. In brief, the corneas were preincubated in Cal-520 AM and CellMask™ Deep Red (Thermo Fisher, Waltham, MA) for 30 minutes at 37°C and 5% CO2. Cal-520 AM was used at 1 : 100, and CellMask Deep Red was used at 1 : 10,000, and the final concentration of DMSO and pluronic acid was 1% (v/v) and 20% (w/v), respectively. Excess dye was removed, and the corneas were mounted in polyethylene glycol on silanized glass bottom dishes [26 (link)]. For in vitro imaging, human corneal epithelial (HCE) cells [17 (link)] were cultured to confluence on glass bottom dishes and preloaded with the calcium indicator dye. Images were collected every 5 seconds for 45 minutes on a Zeiss Axiovert LSM 880 confocal microscope (Carl Zeiss, White Plains, NY) utilizing the FAST module and AIRYScan.
4
Automated Cellular Wound Healing Assay
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For cellular mobilization experiments on cultured cells, HCLE cells were plated on a p35 MatTek well (MatTek Corporation, Ashland, MA, United States) and grown to confluence. For inhibitor experiments, confluent cells were pre-incubated with either ArC 118925XX (Tocris, Minneapolis, MN, United States), A438079 (Tocris), or 10PanX (Tocris) for 1 h to inhibit P2Y2, P2X7, or pannexin-1 respectively. Cells were pre-incubated with SiR-Actin (Cytoskeleton, Inc., Denver CO, United States) (1:1,000) counterstain for 20 min at 37° C. Excess stain was rinsed. Imaging was performed using a Zeiss Axiovert LSM 880 confocal microscope (Zeiss, Thornwood, NY, United States) utilizing the ×20 objective and the environmental chamber maintained an environment of 37°C and 5% CO2. Images were collected every 5 min for 4 h. FIJI/ImageJ (NIH, Bethesda, MD; http://imagej.nih.gov/ij/ ) region of interest analysis was used to quantify wound perimeter over time and calculate the percent wound closure at each time point. FIJI/ImageJ centroid analysis was used to plot the movement of individual cells both parallel to and perpendicular to the wound to calculate their trajectories.
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