This standard technique produces fluorescently tagged gelatin coated cover slips that are suitable for quantitative invaded podium matrix degradation assays. After coding the cover slips with fluorescently labeled gelatin, plate the cells and culture for matrix degradation. Next, fix and fluorescently stain the cells for inve podia marker proteins acquire the images and quantitate the matrix degradation.
Typical results can quantify the differences inve podia mediated matrix degradation between cells with differential manipulation of select genes or treatment with pharmacological agents. Visual demonstration of this method is critical as preparation of gelatin coated cover slips with homogenous fluorescence is difficult to achieve due to uneven coating of fluorescent gelatin or improper handling of gelatin coated cover slips. Demonstrating the procedure will be research assistant professor Karen Martin postdoctoral associate Mandy Ammer and graduate students Elise Walk, Karen Hayes and Steve Markwell.
To prepare the unlabeled 5%gelatin sucrose stock solution, combine the gelatin and sucrose in PBS and to dissolve completely at 37 degrees Celsius. Then store at four degrees Celsius. Proceed to clean the glass cover slips.
Distribute an individual cover slip into each well of a 24 well plastic tissue culture plate to each well at 500 microliters of 20%nitric acid and incubate for 30 minutes. After removal of the nitric acid solution, wash the cover slips three times with deionized water. Next coat.
Each cover slip with 500 microliters of 50 micrograms per milliliter of polyol, lycine, and incubate for 20 minutes at room temperature. Remove the polylysine solution and wash three times with PBS Poly L lysine coating facilitates even coating and bonding of the overlying labeled gelatin. Place 24 well plates on ice and add 500 microliters of freshly prepared 0.5%glutaraldehyde to each.
Well incubate the plates on ice for 15 minutes. Perform three washes with cold PBS then aspirate all traces of PBS prior to gelatin coating. In the meantime, during the glutaraldehyde incubation reconstitute the Oregon Green 4 88 conjugated gelatin.
As per the manufacturer's protocol, warm it and the unlabeled 5%gelatin sucrose stock solution to 37 degrees Celsius. Then dilute 500 microliters labeled gelatin into four milliliters of unlabeled gelatin. Sucrose pipe 100 microliters of the diluted 4 88 gelatin mixture onto each cover slip.
Using enough gelatin to coat the cover slip without manual spreading. It is important to keep the diluted 4 88 gelatin mixture at 37 degrees during the coating procedure to prevent premature solidification. Also, from this step forward, keep the cover slips in the dark as much as possible to avoid potential photo bleaching.
Once all the cover slips are coated in a single plate, hold the plate at an angle and remove excess gelatin from each well by vacuum aspiration. Then incubate the coated cover slips in the dark for 10 minutes at room temperature. After washing the cover slips three times with PBS add 500 microliters of sodium burrow, hydride, sodium burrow.
Hydride is effervescent and small bubbles will be evident on and around each cover slip incubate for 15 minutes at room temperature to reduce and inactivate residual glutaraldehyde. Remove the sodium bur hydride solution by vacuum aspiration with a quick sweeping motion around the outside of each. Well take care not to pick up any floating cover slips that became detached from the bottom of the tissue culture plate during sodium bar hydride treatment after three PBS washes, incubate the cover slips in 70%ethanol for 30 minutes at room temperature.
Using sterile technique, transfer the cover slip containing plates to a type two a B cell culture laminar flow hood and rinse cover slips three times with sterile PBS the with a sterile needle and forceps. Carefully transfer the cover slips to an empty well of a new 24 well plate equilibrate. The cover slips with the culture medium for the specific cell type being acid.
Now seed the cells directly onto the cover slips after an appropriate incubation time. Process samples for immunofluorescence imaging to mount the samples. Place a drop of prolonged gold anti fade or similar reagent on a microscope slide and to carefully position the inverted cover slip, acquire images for analysis.
This analysis provides the normalized area of matrix degradation relative to the area of cells or the number of cells. It is useful for analyzing entire microscopic fields of view where multiple cells are present that have been collectively treated with irna growth factors or therapeutic agents. For this analysis, images collected at lower magnification sufficient to efficiently collect information about populations of cells.
Open the image in image J.Check the scale information and select the measurements. Choose the area and limit to threshold options. Calculate the area of degradation using the fluorescent gelatin image correct for uneven illumination across the gelatin by subtracting the background threshold, the image to set the upper and lower pixel intensity values.
In subsequent images. Use the set button in the threshold window to set the same threshold for all images as an objective means to select degradation area. Use the analyze particles function to measure the threshold at areas of matrix degradation.
Choose a particle size greater than zero to remove noise from the selection. Show outlines to identify regions of interest. Then check display results and summarize to show measurements if the drawing has specifically outlined all of the areas of degradation.
Copy the total area measurement into a spreadsheet if other objects were selected, such as debris, record only the areas of the relevant regions of interest. Next to calculate the cell area open the foid and stained image threshold of the image to set the upper and lower pixel intensity values so that the edges of the cells are selected in subsequent images. Use the set button in the threshold window to set the same threshold for all images.
As an objective means to select cell area. Use the analyze particles function to measure the threshold of area of the cells. Choose a particle size greater than zero to remove noise from the selection.
Show outlines to identify regions for analysis, check display results and summarize to show area measurements. Do not check. Include holes if there are spaces between cells and a cluster.
Now choose. Okay, copy the area results for relevant regions of interest into a spreadsheet and calculate the area of gelatin degradation per total area of cells. An alternative approach is to normalize degradation area to cell number as determined by counting DAP e stain nuclei.
This is important if manipulations alter the average cell area For this approach, use the nucleus counter plugin to automatically count nuclei. Adjust options to optimize for your images. If nuclei overlap extensively, automatically counting may not provide accurate enumeration In this case.
Manual counting can be facilitated using the cell counter plugin when properly performed. Cover slips are evenly coated with Oregon Green 4 88 conjugated gelatin. Several artifacts can occur during the coating and processing of gelatin coated cover slips.
For instance, uneven coating results from improper covering of the cover slip during the coating process due to poor mixing, manual spreading or partial solidification of the gelatin mixture Scrapes indicate removal of the coated matrix by scoring with needles or forceps during handling. Drying of the cover slip surface during prolonged storage periods can result in a cobblestone appearance. This area reflects photo bleaching of the fluorescent gelatin surface during imaging due to prolonged or high intensity light exposure.
Note how a well-prepared cover slip displays homogenous fluorescence when visualized by microscopy. The resulting thin matrices produced during this procedure provide a sensitive means to evaluate the ability of cells to degrade ECM. For example.
These images capture the invaded podium matrix degradation activity of an OSC 19 head and neck cancer cell. Here cells were plated on an Oregon Green 4 88 conjugated gelatin cover slip and imaged by fluorescent microscopy shown as by both 2D and 3D images. These OSC 19 cells plated on Oregon Green 4 88 conjugated gelatin Cover slips were labeled with R domine conjugated Phin and anti cort actin antibodies visualized with ANOR 6 47 secondary antibody and pseudo colored green invaded.
Podia are evident within the merged image as focal cytoplasmic concentrations of F actin and cortan that overlap with areas of gelatin clearing volume fill. Visualization depicts how the invaded podia penetrate into the ECMA sub volume was created to show an edge inside the cell where inve podia are present. The dorsal edge view demonstrates inve podia inserted into the underlying gelatin.
Ventral edge view shows protrusive in Veda podia and areas of gelatin degradation underneath the cover slip as regions of red present the green matrix. These images show some of the important steps for quantification of normalized gelatin matrix degradation cells within an entire microscopic image. All fluorescent images have been converted to gray scale to better display the red thresholding and region of interest markings.
This image of Oregon Green 4 88 conjugated gelatin shows the dark areas where degradation has occurred. Thresholding, the gelatin image highlights the areas of degradation from which selected regions of interest are measured for area of degradation. This image shows rumine, foid and staining of f actin as expected.
The threshold at actin image highlights the total cell area from which cell areas to be measured are selected. Similarly, the technique can be applied to study images of DAPI stained cell nuclei. This panel demonstrates select steps involved in quantifying fluorescent gelatin degradation by individual cells within a mixed cellular population.
This simple quantification is of a single transfected OSC 19 cell over expressing recombinant cort actin fused to the flag epitope tag. Note how the matrix degradation by transfected cells can also be analyzed within a mixed population of transfected and nont transfected cells. While attempting this procedure, it is important to remember several things.
First, be sure your fluorescent cell markers do not overlap with the color of the fluorescent gelatin. When collecting images, it's important to choose fields based on the presence of cells. Do not preferentially search for areas of degradation as this may bias the data Due to variability among cell lines and responses to various therapeutic agents.
Factors such as seating number degradation time and protein selection for immunofluorescent labeling should be optimized for your particular experiment. After watching this video, you should have a good understanding of how to generate cover slips that are evenly coated with fluorescent gelatin from beta podia mediated degradation assays, as well as methods for quantitation of matrix degradation.
