The data set I'm going to be using is published, it's my own data, it's what I call a Phospho-Flow + Intracellular Cytokine Staining assay, PFICS. This is a polyclonal stim assay where I thawed and rested crypreserved human PBMC overnight, stimulated with a mitogenic cocktail. Here a PMA+ionomycin for two hours or rested some cells as a no stim while blocking protein secretion. And you block protein secretion to allow the cytokines to build up to detectable levels inside the cell so that you can visualize them in a flow assay. After the two hour stim we're going to stain with a viability dye; stain for the surface antigens, in this case the panel is CD3, CD4, and CD8 to identify major T-cell subsets. CD38 and HLA-DR, that's class 2HLA. These will identify activated cells in the T-cell fractions and are important markers of immune inflammation in HIV disease and other retroviral infections.
After I do the surface staining, I'm then going to stimulate with PMA+ionomycin for another 20 minutes or rest as a no stim, then fix permiablize and stain for the intracellular antigens. My readouts in this assay are gonna be the phosphorylation of ERK1/2, that's the extracellular signal-regulated kinase protein. Type 2 interferon, interferon gamma and Perforin, which is a cytotoxic granule-associated protein involved in poking holes in the membranes of virally-infected cells to deliver cytotoxic granzymes.
When you have two different stim conditions like this, you actually have four potential combinations of stimulatory environment that these cells can see, which turns the assay into a simple time course illustrating the differential kinetics between the phosphorylation response of the intracellular signaling intermediate and the type 2 interferon response. That's the cytokine that gets produced in response to the signal. If I don't stim the cells at all, I'm not gonna see much phosphorylation or interferon gamma. If you do a short, 20-minute stim, you get a whopping phosphorylation response, shown here in blue, but that's not sufficient for interferon gamma to come up to detectable levels inside the cell. If you do a two hour or two hour and 20 minute stim, however, you get both signaling and a cytokine response in these cells. These are overlaid histograms and I'll show you how to make these in a few minutes in the layout editor where we put four different stimulation conditions, so four different, individual samples on the same plot so that we can illustrate a response in this time course under these different conditions.
I'm gonna load 46 samples. 20 of these are my real experiment. I call these All Stain samples where you stain with all the reagents in the panel. And 14 are FMO controls. These are Fluorescence Minus One control where you leave one reagent out of the panel and it serves as a gating control to allow you to see where the true negative is in the context of all the other colors in the panel and it serves as a guide for where you can draw your gate for positive and negative. Over here on the left I have an All Stain. We're looking at CD3 versus HLA-DR. You've got your T-cells on the right. Your antigen-presenting cells, like B-cells and monocytes on the top left. And then the double negatives like natural killer cells are gonna be found in the bottom left corner.
If you don't put any HLA-DR antibody into your staining reagent panel, then you get a nice, clean DR signal and this would guide me where I would place a gate to define the positivity for HLA-DR. Then I also have these compensation controls and we may not get into compensation today. Compensation is required for multicolor fluorescent cytometry and you isolate the fluorescent spectrum for each reagent that you're using in your panel so you can determine the spillover into different detectors and then correct for that. Here is a uncompensated sample where I've stained just with the CD3 Alexa700 antibody. You can see the skewing of the Alexa700 signal into the APCH7 detector. And if I were to compensate this control, it would look more like this where the positive and negative fractions become equal in their medians in the spillover channel.
I do have some other webinars that we have done. I do one called Ninja Skills where I start with compensation and I can point you to those resources here at the end.