The overall goal of this procedure is to visualize vascular calcium signaling triggered by pericrine derived reactive oxygen species or r os. This is accomplished by first visualizing R os using oxidation sensitive dyes in active macrophages. ROS can also be visualized in endothelial cells using plasmid based fluorescent sensors.
Then proceed to simultaneously visualize cell tracker red labeled activated macrophages and flu of four labeled endothelial cells in a co-culture model. Finally, measure the calcium signaling in endothelial cells triggered by macrophage derived R os. By using a confocal imaging system.
These results can demonstrate R os levels independently as well as simultaneously assess intracellular calcium mobilization triggered by Peregrine derived ROS. This method enables simultaneous assessment of two different signaling molecules that intimately elicit a vascular signaling and simulator pathophysiological setting. Two postdocs in my laboratory, Karthik and Raj will demonstrate the procedure on raw and calcium measurements and macrophage endothelial cell interactions.
Culture mouse monocyte derived macrophages on glass bottom 35 millimeter dishes. Then challenge the cells with toll-like receptor agonists and incubate for six hours at 37 degrees Celsius. For visualization of the cytosolic.
ROS add oxidation sensitive dye and incubate the cells for 20 minutes. Now visualize the cells on a temperature controlled stage of a confocal imaging system. Alternatively, to visualize mitochondrial ROS in TLR activated cells, add the mitochondrial superoxide indicator and to incubate the cells for 30 minutes at 37 degrees Celsius.
For a negative control, treat the cells with either PBS or DMSO for a positive control. Treat the cells with two millimolar antimycin. A proceed to visualize all cells on the temperature controlled stage of a confocal imaging system.
Using image J software, analyze all the image data. The fluorescent sensor hyper specifically senses sub micromolar concentrations of peroxide and based on mammalian expression, vector design can be targeted to either cytoplasm or mitochondria. Using electroporation, create stable lines of mouse pulmonary microvascular endothelial cells bearing inappropriate hyper expression plasmid.
To facilitate colony growth plate cells at low density in complete TMEM and incubate for 48 hours, replace the medium with complete culture medium supplemented with a selection antibiotic. Screen clones for the highest percentage of hyper positive cells and amplify the culture to create stable hyper positive lines in individual experiments. Seed cells on 0.2%gelatin coated cover slips and culture overnight to 80%confluence.
Now activate the cells with TLR ligands and incubate for six hours at 37 degrees Celsius. For the detection of cytoplasmic ROS, mount the cover slips of cells onto an adelo cell chamber. Add one milliliter of prewarm tanks, balance salt solution and placed the chamber on a temperature controlled stage of the confocal imaging system.
Record fluorescence changes in cells for the detection of mitochondrial.ROS. Include a positive control of two micromolar antimycin A.Then prepare the cover slips of treated cells for confocal microscopy. Perform image analysis using image J software.
Start with endothelial cells grown to 80%confluence on gelatin coated glass cover slips for the visualization and measurement of intracellular calcium changes. Incubate cells in the fluorescent dye flu oh 4:00 AM at room temperature for 30 minutes to minimize dye loss wash cells with experimental imaging solution containing suen pyro zone. Now mount cover slips onto an adelo cell chamber at 800 microliters of pre warmed experimental imaging solution and position the cells on a temperature controlled stage of the confocal imaging system in a separate tube.
Use 500 nanograms per milliliter of cell tracker red C-M-T-P-X for 15 minutes to label the macrophage population under investigation wash cells and resuspend pellets in 200 microliters of experimental imaging solution. Now add the labeled macrophages onto the flu oh 4:00 AM loaded. Endothelial cells in the confocal chamber assembly record the green and red fluorescence every three seconds for 10 minutes.
In this experiment, J 7 74 0.1 cells were challenged with TLR two, three, and four ligands for six hours. Representative confocal images show an increased cytosolic R os as measured by DCF. Interestingly, mitoses socks red fluorescence indicates a similar increase in mitochondrial ROS.
These fluorescence changes are quantitative. Thus results must be normalized and expressed as relative fold change per treatment, the antimycin a signal validates a positive control for mitochondrial ROS production. This figure shows images of endothelial, cytosolic and mitochondrial peroxide levels in stably expressing hyper cyto or hyper D mito.
MP ECS stimulated with TLR agonists. Quantitation of mean fluorescence change demonstrates increase of hyper cyto and hyper D mito fluorescence after six hours of stimulation in a co-culture system. To assess Peregrine ROS signaling freshly isolated mirin macrophages from both wild type and GP 91 Fox null mice were activated by one microgram per milliliter of LPS for six hours.
Macrophages were labeled red with C-M-T-P-X and added onto green flu four.Loaded. MPM ecs. Interestingly, LPS activated macrophages isolated from wild type mice triggered large and rapid calcium mobilization in MPM ECS compared to macrophages from GP 91 Fox null mice.
While LI attempt take this procedure, it is important to remember that either prolonged downloading or laser exposure lead to auto oxidation of DCF. It is crucial to avoid photo bleaching of hyper cyto and hyper D mitral. Once mastered RA measurement in macrophages as well as in endothelial cells can be done in seven hours if it is performed properly.
