Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

The max Cytokine secretion assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable cytokine secreting cells in order to label IL 17 secreting cells. A single cell suspension of mouse leukocytes is prepared and stimulated at 37 degrees Celsius with PMA ion mycin to induce cytokine secretion to stop secretion. Cells are then placed on ice where they are exposed to the IL 17 catch reagent.

Once secretion is reinitiated by increasing the temperature to 37 degrees Celsius, IL 17 is trapped by the catch reagent. A bispecific antibody that binds to CD 45 on the cell surface of leukocytes and to IL 17 as it is secreted and caught near the cell surface secretion is then stopped again by placing cells on ice to detect the trapped IL 17 cells are incubated with a second IL 17 specific antibody conjugated to biotin And an anti biotin PE antibody cells can now be directly analyzed By flow cytometry or prepared for isolation and enrichment by subsequent labeling with anti PE conjugated microbeads. Hi, my name is Ellie Rankin, one of the scientists at Mill Tenney Biotech.

Today I'm going to show you a procedure how to isolate and detect TH 17 leukocytes, which upon stimulation secrete IL 17 IL 17 plays an important role in the adaptive immunity and inflammatory response and is also important in the driving the autoimmune inflammation. So let me show you how the assay is done. For today's Protocol, we need to first make a buffer containing PBS with BSA and two millimolar EDTA because air bubbles can block max separation columns, the buffer needs to be degas and stored at two to eight degrees Celsius before use.

Second, we use RPMI 1640 medium containing 5%mouse serum. The culture medium should not contain BSA or FCS as these compounds will alter the specificity of cell stimulation. Lastly, you will need the mouse IL 17 secretion assay cell enrichment and detection kit from MIL 10 E Biotech.

The kit contains the following components, the IL 17 catch reagent, the IL 17 detection antibody, biotin, the anti biotin pe, and the anti PE microbeads. Logically You will likely carry out this protocol with a negative and positive control such as an unstimulated, PLE cytes and a counterstain for T cells. For our demonstration, we will focus on the stimulated cells only.

This protocol will be carried out using sterile technique. Our protocol begins with a single cell suspension of mouse SP cytes isolated using the gentle max associator. The concentration of cells should be predetermined via cell counting.

Pellet the cells at 200 Gs for 10 minutes at room temperature Following centrifugation, we aspirate the super natin off the pellet using a pipette. Do not decant the tube to avoid loss of the pellet. Now the cells should be resuspended in our culture medium and then add it to the, well add sufficient medium for a concentration of 10 million cells per milliliter and 5 million cells per square centimeter.

To stimulate an immune response in our resuspended cells, we add ion mycin and PMA to the sample and mix the solution by gently pipetting up and down. Then the wells are labeled accordingly. Now we will incubate ourselves for three hours at 37 degrees Celsius with no mixing to start the stimulation period.

Proceed to IL 17 analysis three hours from the onset of stimulation. So plan accordingly. Here we see stimulated mouse spleen cells.

Please note the distinct morphology and cell clustering of stimulated cells. For clarity, we will follow only the stimulated cells. From here on to stop secretion, we collect the stimulated cells by gently pipetting up and down.

With cold buffer cells are then transferred from the well to a tube and are washed a second time. To ensure that all cells are collected, it's a good idea to check your dish under a microscope. If cells still remain attached, you can collect the remaining cells by rinsing the dish.

With cold buffer, any cell clumps in your cell suspension can be removed using the pre-participation filters. One potential Pitfall of this procedure is cross-contamination of the catch reagent, which can occur during labeling. When the bispecific antibody binds to a nons secreting T-cell and traps IL 17 secreted from a neighboring lymphocyte, thereby generating false positives in order to circumvent this problem, it is critical to cool cells down prior to labeling and work with cold buffer to limit secretion and slow diffusion of IL 17 and avoid this cross contamination.

It is paramount to note that cross-contamination can also occur if greater than 2%of IL 17 secreting cells are present. If the concentration of IL 17 secreting cells is expected to be greater than 2%adjust volumes accordingly. Hence, in addition to keeping cells cold, they must be kept at a defined concentration.

To begin the labeling procedure we use 10 million cells in a 15 milliliter closeable tube. If higher cell numbers have to be used, simply scale up all volumes accordingly. Once the optimal cell concentration has been obtained, wash the cells by adding 10 milliliters of cold buffer.

Now spin down the cells at 300 Gs for 10 minutes in a refrigerated centrifuge following centrifugation, aspirate the supernatant completely using a pipette. Do not decant the supernatant as this will lead to cell loss and imprecise volumes. Repeat the washing step which consists of adding 10 milliliters of cold buffer centrifugation and aspiration.

Now that we have a pellet of desired purity, resuspend the cells in any microliters of cold culture medium to label them. We will now add 20 microliters of mouse IL 17 catch reagent. Incubate the cells for five minutes on ice.

After the five minute incubation period on ice, remove the tube and dilute the cells in 10 milliliters of 37 degrees Celsius. Warm medium. Then secure the tube on the max mix tube rotator and incubate the tube at 37 degrees Celsius for 45 minutes.

Under continuous motion, increasing the temperature to 37 degrees Celsius will restart cytokine secretion Following the 45 minute secretion period at 37 degrees Celsius. Place the tube immediately on ice. This will stop cytokine secretion.

From here on it is critical to keep cells on ice. Working with cold buffer will avoid cross-contamination of the catch reagent. Fill up the tube with cold buffer and centrifuge at 300 Gs at two to eight degrees Celsius for 10 minutes.

Aspirate the superber natin completely. Repeat the wash Step one more time. The Cell Pellet is resuspended in 80 microliters of cold buffer and the tube is placed on ice.

To avoid non-specific binding of the antibody, we recommend the addition of 10 microliters FCR blocking reagent and incubate on ice for five minutes. Then we add 20 microliters of mouse IL 17 detection antibody that is conjugated to biotin and incubate for 10 minutes on ice. Once again, wash cells as we have done before by adding 10 milliliters of cold buffer and centrifuge at 300 Gs at two to eight degrees Celsius for 10 minutes.

Now aspirate and resuspend the cell pellet in 80 Microliters of cold buffer and add 20 microliters of our secondary antibody. Anti biotin pe. Mix the cells and secondary Antibody solution and again incubate the tube for 10 minutes on ice.

Once the second incubation is complete, wash the cells with 10 milliliters cold buffer and centrifuge. The pellet can now be resuspended in 500 microliters of cold buffer. If the cells have to be separated for further downstream analysis, they can be magnetically labeled with anti PE microbeads and separated manually or automated using max columns and max separators.

We have now completed the labeling of our SPLU cytes and Are ready for analysis for our analysis. We will compare the stimulated and unstimulated SP cytes by flow cytometry before running. Facts cells are stained with propidium iodide at 0.5 micrograms per milliliter to gate out dead cells.

The flow cytometer was loaded with 200, 000 cells for each sample and we now are going to look at the scatter plots of relative antibody reactivity in the samples. Two important considerations for analysis of rare cells like IL 17 positive cells by flow cytometry are setting a gate on lymphocytes in the forward scatter versus side scatter plot and gating out dead cells stain with propidium iodide and B cells which may cause unspecific background staining. In order to further enhance the sensitivity of detection.

Here we use CD four A PC antibody to detect T cells in the CD 45 RB two 20 per CP antibody. To detect the B cells, we see the forward and side scatter properties of the samples and apply a gait on the lymphocyte population relation. A second gate was applied to see the stained dead cells and stained B cells on the Y axis.

These cells are excluded from the T cell analysis. In this example, which is essentially our negative control, we can see that there are very few IL 17 secreting CD four positive T cells in unstimulated samples approximately 0.003%Looking at the stimulated T-cell population, we can see a substantial number of CD four positive T cells that secrete IL 17, about 0.367%before separation and 60 point 76%after magnetic separation with max technology. We've Just shown you through video documentation how to use mul 10 biotech's mouse IL 17 secretion assay for the cell enrichment and detection kit using pe.

The most important part of this procedure is to have an idea of the frequency of IL 17 secreting cells that you have in the starting population. The correct cell concentration will ensure that the labeling and the cytokine secretion period has less cross-contamination, so ensuring reliable results. So that's it.

Thanks for watching and good luck with your experiments.