The overall goal of the following experiment is to assess the induction of cellular new one sase activity in response to ligand binding to its specific receptor. This is achieved by first culturing cells on 12 millimeter circular glass slides in a 24 well tissue culture plate for 24 hours. As a second step, the live cells adhered to the glass slide are placed directly in contact with a previously prepared mixture of sase substrate and receptor ligand with or without specific inhibitors on a microscope Slide next.
The microscope slide is immediately placed under a fluorescent microscope in order to visualize and digitally capture the fluorescent images. Over time results are obtained. That show changes in new one sase activity on the cell surface associated with the ligand treated live cells based on the blue fluorescence intensity surrounding the cells under epi fluorescence microscopy.
The main advantage of using this technique over existing methods like spectra photometric analysis is that you use less reagents. You can also observe and capture images of sase activity in live cells. This method can be used to answer key questions in the immunology field, such as looking at potentially novel ligand receptor interactions, and also looking at the effects of specific enzyme or protein inhibitors on receptor activation.
Demonstrating the procedure will be Dr.Ray Amit and Dr.PT Gath, former PhD students from my laboratory who use this assay in their research studies Prior to resurrecting frozen macrophage cells. Prepare culture medium using sterile filtered delcos, modified eagles medium supplemented with 10%fetal bovine serum, and five micrograms per milliliter of the antibiotic plasmin. Then add four milliliters of the culture medium to a 25 square centimeter cell culture flask for every vial of frozen cells, remove the vial of frozen cells from the minus 80 degrees Celsius freezer and quickly thaw by hand warming in the sterile biohazard containment hood.
This will take about two to three minutes. As soon as the cells are thawed, use a one milliliter sterile pipette to transfer them quickly into the cell culture flask containing the conditioned medium. Place the flask containing the cells in a tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide using an inverted microscope.
Check the cells daily for growth and adherence until they become 75%to 90%confluent a few days prior to plating cells for the SASE assay. Prepare the 12 millimeter circular glass slides in which the cells will be plated. In the biohazard containment cabinet.
Place slides in a large glass Petri dish and sterilize them by submerging in 100%ethanol for one hour after one hour. Remove the alcohol and leave the wet glass slides exposed to the sterile filtered airflow in the cabinet under UV for 24 hours or until they're dry to plate. The cells first sterilize a full curved four inch serrated stainless steel forceps by flaming.
Then use the forceps to carefully place a single sterile circular glass slide into each well of a 24 well flat bottom tissue culture treated polystyrene plate. Using a five milliliter sterile pipette, remove the DMEM conditioned medium from the flask with the adherent macrophage cells. Wash the cells once with one x sterile tris buffered saline pH 7.4 to remove any serum containing medium.
Next, add one milliliter of calcium medium free phosphate buffered saline at pH 7.4. Place the cells in the 37 degree Celsius incubator for approximately 10 minutes or until the cells start to lift off in the flask. Once the cells have lifted off, add three milliliters of DMEM conditioned medium to the suspended cells.
Then transfer 0.5 milliliters of the suspension with approximately 0.5 million cells to each 12 millimeter circular glass slide in the 24 well plate incubate the tissue culture plate containing the macrophage cells at 37 degrees Celsius in 5%carbon dioxide for 24 hours to allow the cells to adhere to the circular glass slides. The assay for sase activity will be performed under three different conditions on three separate microscope. Slides on the first slide, which serves as a negative.
Control cells are exposed to mounting medium. The neuraminidase substrate for ANA and tris buffered saline, but not the toll-like receptor ligand LPS. On the second slide, cells are exposed to mounting medium form ana the toll-like receptor ligand LPS and tris buffered saline.
On the third slide, cells are exposed to mounting medium form ANA, LPS Tris, buffered saline, and the neuraminidase inhibitor Tamiflu. We will demonstrate the procedure for just the second assay since the three assays differ only by the mixture of compounds on the microscope slide. Place the microscope slide on the lab bench and add the following compounds in sequential order.
One microliter of mounting medium followed by one microliter of form two microliters of LPS and lastly, three microliters of tris buffered saline, pH 7.4. Next, remove the medium from one well of the 24 well plate containing the cells. The next step is the single most difficult aspect of this procedure because you have to remove the circular glass cover slip from the well of the tissue culture plate with the forceps without breaking it With four inch forceps.
Gently lift the circular glass slide to the corner of the well and then carefully remove and place the slide with the cells facing down in the solution mixture. On the microscope slide, take the microscope slide immediately to an epi fluorescent microscope that has been set for the UV filter and equipped with a digital camera for capturing fluorescent images. Focus the microscope under phase contrast until you can see the cells and then switch to fluorescent mode.
Acquire images of the cells under fluorescent mode at one minute, two minutes, and three minutes. In addition, acquire a single image of the cells under phase contrast representation. Results of a sase assay experiment are shown here.
First, as a negative control, live cells adhered to a glass slide. Were placed in direct contact with a spot mixture of mounting media and form substrate IP sase activity were present. The substrate would be hydrolyzed to produce free ethyl lum beone, which has a fluorescent emission at 450 nanometers following excitation at 365 nanometers taken at two minutes after adding substrate.
This fluorescent image shows no sase activity surrounding the cells. This next image shows the results from a positive test SASE assay in which live cells adhered to a glass slide were placed in direct contact with a spot mixture of mounting media four substrate and TLR four ligand LPS. The substrate was hydrolyzed by sase enzyme to produce free ethyl lum beone, and the fluorescent image taken at two minutes after adding substrate and LPS indicates high levels of sase activity surrounding the cells.
However, when live cells were placed in direct contact with a spot mixture of mounting media form nano substrate TLR four ligand LPS, as well as the neuraminidase inhibitor, Tamiflu sase activity was greatly reduced or no longer detected depending on the concentration of the inhibitor. Finally, this graph shows the 50%inhibitory concentration of Tamiflu and Oseltamivir carboxylate determined by plotting the decrease in sase activity against the log of the agent concentration, the mean fluorescence surrounding the cells for each group was measured. Using Image J software While attempting this procedure, it is important to remember to culture the cells on circular glass cover slips in 24 well tissue culture plates 24 hours prior to starting the Sade assay.
In addition, all the reagents that are used in this assay must be prepared prior to starting the assay.
