The overall goal of this procedure is to detect T-cell responses to antigens of interest. This is accomplished by first coding with capture antibodies. The second step of the procedure is to add cells and antigens of interest and then incubate overnight.
The third step of the procedure is to add detection antibodies. The final step of the procedure is developing the plate with substrate reagents and analyzing spots. Ultimately, results can be obtained that chose cytokines of interest through analysis of spot forming units.
Hi, my name is Dr.Ki Richter and I'm a research instructor in Dr.Wonder Drake's Laboratory at Vanderbilt University in the division of Infectious Diseases. Today we are demonstrating the Ellie Spot technique, also known as the enzyme linked immuno spot assay. Dr.Isfahan chambers and Tiffany Cone will be demonstrating the technique for you today.
In order to maintain cell culture standards, perform manipulations under sterile conditions in the hood, open an ally spot plate and wash three times with PBS. Prepare a coating solution of the capture antibody in PBS. Mix well by vortexing for convenience.
Transfer the antibody solution to a reservoir for the multichannel, pipetter and aliquot 100 microliters of coating solution into each. Well seal the plate with perfil and place in a refrigerator overnight. These antibody coated plates are good for weeks.
The rule of thumb dictates that a plate can be used if there is still liquid left in the wells. To prepare the cells for this assay stain freshly isolated peripheral blood mononuclear cells with trien blue for viability and count them Plate the pbmc at 2 million cells per milliliter in our 10 media and incubate overnight at 37 degrees Celsius with 5%carbon dioxide. In order to keep track of the experiment, label the Ali spot plate lid with the specimen identification number, well conditions and date.
Wash the plate six times with sterile PBS using the dump and blot method. Be careful not to splash the plate while dumping, as this could cause the wells of the plate to turn purple. Add our 20 media to each well and incubate the plate at 37 degrees Celsius for one hour.
While the plate is incubating, count the cells that were rested overnight. Harvest the pbmc by centrifugation. Decant the supernatant and resuspend the pellet in our 10 media at a concentration of 1 million cells per milliliter.
After the one hour incubation of the ally spot plate, remove the R 20 media using the dump and blot method. Be careful not to splash the plate while dumping. Add 100 microliters of cell suspension to each.
Well follow the experimental design with addition of peptides and antigens to the corresponding test wells. Always include a negative control of cells with no peptides and also positive control wells with added phyto hemagglutinin. Incubate the plate overnight at 37 degrees Celsius with 5%carbon dioxide.
Wash the plate six times with 150 microliters of one XPBS using the dump and blot method. Add 100 microliters of PBS to each well of the plate and place the plate in the refrigerator or at room temperature for 15 minutes. Meanwhile, prepare the biotin antibody solution in PBS.
Take the ALI spot plate out of the refrigerator and discard the PBS by flicking it into the waste basket. Be careful not to splash the plate while dumping a aliquot the biotin antibody solution into each well and incubate the plate in the tissue culture hood at room temperature for one hour. Wash the plate six times with PBS using the dump and blot method on the last wash.
Leave the PBS on the plate now. Prepare the STREPTAVIDIN antibody solution in PBS, making sure to vortex the solution to mix. Well discard the last PBS wash from the wells and plot the ALI spot plate.
Add streptavidin antibody solution to each well and incubate the plate in the tissue culture hood at room temperature for one hour. In order to remove excess trevain antibody, wash the plate six times with PBS using the dump and blot method on the last wash, lead the PBS on the plate. Given the light sensitivity of the B-C-I-P-M-B-T alkaline phosphatase substrate solution.
Work in the dark when preparing the stalk solution as described by manufacturers in the alkaline phosphatase substrate kit instructions discard the remaining PBS from the wells and blot the ly spot plate. Add substrate solution to each well and turn on the light. After five to 10 minutes of color developing, allow the reagents to remain on the plate until the spots begin to turn purple and are quite dark.
This takes up to 20 minutes. Wash the Ali spot plate three times with tap water and allow to air dry. Each plate contains a positive control, negative control and peptide of interest conducted at the minimum in duplicate.
The positive control wells are a deep purple color reflect a confluent layer of cells and hardly any white background. The negative control wells have no purple background and no spots as expected. The test sample wells illustrate cellular responses to microbial peptides here.
The purple spots represent a single responsive cell expressing the cytokine of interest When conducting the LA spot technique. Please be careful to not puncture the membrane with your pipette tips or to allow your plate to dry out because this will lead to high background.
