Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

The overall goal of this procedure is to study the mechanisms that regulate mast cell, secretory granule development and cytosis. This is accomplished by first cot transecting mast cells with a reporter gene for exocytosis, as well as a gene of interest. The second step is to assess the effect of the tested gene on exocytosis by stimulating the cells and monitoring the fluorescent signal of the reporter gene during exocytosis.

Next, the effect of the tested gene on mast cell secretary granule generation and development is assessed by confocal imaging of the reporter gene that is localized to the secretary granules. The final step is to perform morpho metric analysis of the secretory granules. Ultimately fluorescence labeling of mast cell secretory granules using a reporter gene is used to show alteration in mast cells, secretory granule development and their exocytosis.

The the main advantage of this technique over existing methods, such as measuring the release of the endogenous mediators, beta haase, or histamine, is that it is based on the co-expression of the gene of interest while type mutant or S-H-R-N-A together with a reporter for exocytosis. The co-expression then allows monitoring secretion exclusively from the genetically manipulated cells, thus overcoming the problem of high noise to signal ratio that arises when measuring the release of the endogenous mediators due to the low transfect ability of mast cells, which hampers the interpretation of the results. This method can help answer key questions in the field of the cell biology of mast cells, such as identifying the molecular mechanisms that control the process of secretory granules generation, including calgo selections, packaging of the granules, and their exo cytosis competence.

This method also helps in identifying the molecular mechanisms that control the process of mass cells regulated exo cytosis by assessing the involvement of kinases, phosphotases, and other family of proteins in this process. To begin, prepare rat basophilic leukemia cells and media as described in the text protocol to perform transfection. First, remove the culture medium from the plate using a sterile pasture pipette connected to a vacuum.

Rinse the cell monolayer with prewarm trypsin, A DTA that covers the whole monolayer. Place the plate in an incubator at 37 degrees Celsius for no more than 10 minutes. Then check if the cells have detached using the optical microscope.

Once detached, add medium to the culture plate to suspend the cells at a volume of at least two times the volume of the trypsin solution. After counting the cell number, using a hemo cytometer pellet, 15 million cells by centrifugation for three minutes at 200 G and 25 degrees Celsius. Discard the SNAT and add 280 microliters of transfection medium.

Then transfer the reaction mixture into a four millimeter vete. Add 20 micrograms of plasmid containing neuropeptide Y-M-R-F-P, also known by its short name N-P-Y-M-R-F-P, and either 30 micrograms of control, empty plasmid or a tested plasmid. The final volume of the reaction mixture should be 300 microliters.

After placing it on ice for 10 minutes, wipe the vet free of residual water. Then proceed to electroporation at 300 volts for nine milliseconds. For measurements of exocytosis, reflate the cells immediately in 24, well tissue culture dishes containing 300 microliters of medium.

Add eight microliters of the reaction mixture to each. Well then add nont transfected cells to additional wells for the control. Prepare enough wells to have duplicate wells for each treatment, for every transfection for time-lapse microscopy reflate the cells immediately in an eight well chamber BO silicate cover glass system containing 80 microliters of medium.

Add 1.5 microliters of the reaction mixture to each chamber to sensitize the cells for FC epsilon R one mediated activation. Add one microgram per milliliter of mouse DNP specific monoclonal IgE to the media. Incubate the cells with IgE for at least two hours.

After 18 to 24 hours, use a fluorescent microscope to confirm that the cells express the plasmids. MRFP has an excitation wavelength of 584 nanometers and an emission wavelength of 607 nanometers. N-P-Y-M-R-F-P should appear in vesicular structures which correspond to the secretary granules.

To measure N-P-Y-M-R-F-P exocytosis, remove the culture medium. From the 24 well plate and washed three times with tyro buffer. Prepare unstimulated cells by adding 200 microliters of tyro buffer to the control wells.

Then add the activating reagents, diluted tyro buffer before incubating at 37 degrees Celsius for 30 minutes. Carefully transfer the S supernatants of each well to a 96 well plate. Place the plate on ice and protect from light.

The S supernatants contain the chimeric peptide N-P-Y-M-R-F-P that was released from the cells. Next, add 200 microliters of tyro buffer containing 0.5%Triton X 100 to each well and incubate at 37 degrees Celsius for 10 minutes. This step is important for preparation of cell lysates that contain the remaining N-P-Y-M-R-F-P that was not released from the cells.

Collect the cell lysates and transfer to a 96 well plate before placing on ice. In the absence of light, measure the fluorescence of the cell supernatants and cell lysates using a fluorescence plate reader with a 590 nanometer 20 nanometer bandwidth excitation filter and 635 nanometer. 35 nanometer bandwidth emission filter seed 1.5 microliters of transfected cells in an eight chambered BO silicate cover glass system.

Incubate the cells for 18 to 24 hours in an incubator at 37 degrees Celsius. Remove the culture medium from the chambers and wash three times with tyroid buffer. Then add 72 microliters of tyroid buffer to each chamber.

Use a confocal fluorescence microscope equipped with a heated chamber carbon dioxide controller and a 40 x or 63 x objective. Turn on the microscope systems, including the mercury lamp, computer and lasers. Make sure that the heated chamber is at the right temperature.

Before starting the experiment, place the eight well chamber bore silicate cover glass system into the heated chamber and make sure that the chamber is installed correctly and is stable. Turn on the fluorescence light according to the relevant floor four and visualize the transfected cells turn off fluorescence once a cell of interest is in the field. In order to minimize bleaching and cytotoxicity, this field should contain about two to three transfected cells so that they are well spread but not touching each other.

In this case, the cells are cot transfected with the reporter gene, N-P-Y-M-R-F-P and A GFP tagged constitutively active RAB five mutant. Adjust the laser power to minimize noise and oversaturation as well as toxicity, and set the gain and offset to modify the signal to noise ratio. Scan fast in order to minimize the duration of laser exposition.

Next, set the parameters for the Zack to reconstruct the image in three dimensions. Adjust the pinhole size to one area unit to give the best signal to noise ratio and acquire successive scanning of two dimensional confocal optical slices in the Z axis with optical slices, less than or equal to 0.7 micrometers. Now set the interval time between each acquisition to 30 seconds and the duration of total acquisition to 15 minutes and start acquiring images after five minutes of acquisition.

Pause the time series, add eight microliters of the 10 x trigger and continue the acquisition immediately. Finally, save the pictures. Perform deconvolution using deconvolution software and reconstruct the stacks to three dimensional images and a movie To perform image analysis.

Import the data from the de convoluted time series images that were obtained by confocal fluorescence microscopy to scientific image analysis software such as MRS. This software reads more than 40 microscopy files. To add time points to the end of an open data set, select edit add time points and import the new data set.

Adjust the contrast and color shade to get the optimal image view. The files that were downloaded create the surface of the MRFP channel. Using the surface wizard option.

Choose the default algorithm and the channel N-P-Y-M-R-F-P mark the smoothing option to reduce noise. To avoid loss of small details, reduce the area detail level to between 0.05 and 0.1 microns. Next, set the intensity threshold.

A new gray surface is displayed. Then go to settings and switch the style from center point to surface. Go to the statistic tab in the properties of the selected object.

Then select the detailed tab and the specific value such as fluorescence, intensity or volume. Review the data and confirm that the values are compatible with the images. For example, two or more adjacent granules might be measured as one bigger granule for quantifying the average granule size.

Divide the size of the merch granules to the actual number of granules. As a final step, export the desired data sets to excel files and analyze the data. Shown here are representative time-lapse images of mast cell giant secretory granules that were formed by expression of constitutively active RAB five, as well as the kinetics of exocytosis and the resolution of a single secretory granule.

After stimulation. During mast cell exocytosis, the secretary granules move towards and fuse with the plasma membrane. The granules are coated by constitutively active RAB five shown in green.

The secretary cargo, which is N-P-Y-M-R-F-P in this experiment and is colored in magenta, is then released from the cells into the extracellular space. The granules that were fused by the constitutively active mutant of RAB five shown in green release. Their cargo N-P-Y-M-R-F-P that in this experiment is colored in magenta from the cells into the extracellular space.

The release of N-P-Y-M-R-F-P during exocytosis results in a dramatic increase in N-P-Y-M-R-F-P fluorescence intensity and the shrinkage of the N-P-Y-M-R-F-P containing secretory granules followed by their disappearance, fluorescent intensities of secretory granules. After stimulating the cells with a calcium ionophore are shown, the secretory granules undergo exocytosis at different time points. While one secretory granule did not fuse with the plasma membrane and its fluorescence intensity remained constant.

After watching this video, you should have a good understanding of how to monitor ma cell secretory granules during the development and exocytosis using a fluorescent gene reporter that is delivered to the secretory granules and is released by stimulated cells. Together with the endogenous mediators, The mechanisms underlying mast cell, secretory granules development, and exocytosis can be identifying using this procedure by co-ran, inspecting the cells with a report gene and a tester gene. The role of the tester gene on mast cell secretory granules development can be assessed by performing a morphometric analysis of the secretory granules of the cells that are co transfected with the gene of interest as compared to control cells.

In addition, the role of the tested gene on regulated exo cytosis can be identified by monitoring the release of the reporter gene.