I would first off rename the label Axis with what you are looking at (for example CD3-PE instead of just PE). Not sure what is up with FITC it looks like you do not have Bi-Exponential display on. It is not on for any of the fluors. I would either display as offset graphs of the change with histograms or do Median Fluorescent intensity on those fluors. I can't really help you on much more but depending on where you see the changes percent of platelets or change in intensity and the way the experiment was set up length of exposure versus addition of inhibitors. you have other options too. If you need help I would be more than happy to thru zoom go over a few items let me know.

Hello John, thank you for the offer and tips, do you have any instruction or sources regarding setting up the analyzer? John Powers

For Setting up the BD LSRFortessa™ X-20 Cell Analyzer does it use DIVA?

If so, I first adjust voltages of the detectors to minimize background (erroneous off target signals as well as negative population). I then do Area Scaling. https://www.youtube.com/watch?v=VqNmgwZrJ3M.

To check to ensure that Area correlates to height of signal and I do not lose resolution.

For analysis, my layouts start with (1) a general gate for the area where my cells od interest should be (low granularity/small cells or High Granularity/Large cells (2) FSC-A versus Width or Height to eliminate doublets. (3) Then I do a second pass (Usually unneccessary) but SSC-A versus Width or height. (4) I then do my viability staining versus FSC.

I start to get more stringent these cells at this point should be the best to analyze from. Now i start using either my dump gates or my markers of interest to tweeze out my populations.

I would suggest also https://expert.cheekyscientist.com/technical-programs/ and also https://learning.isac-net.org/

These programs would be good in the long run but some of this info is hard to get across by typing and is easier to show.

John Powers thank you very much for the information, I will use it dearly. What is your opinion on using more than two markers, as in more than lets say FITC and PE, perhaps add APC, BV... etc etc ?

How would you handle compensation if that needs be done ?

Aleksandar Dishkelov Alot goes into development of a good flow panel. I think alot of people do not spend enough time doing it or validating it. I can put tips or info here but it is not the same.

With that I would use whatever markers are going to give you the highest resolution of your cell of interest. like the Greek saying Know thyself however in flow it is "Know thy cell".

We tend to go with at least 8 in total to either omit or compliment the cells that we are targeting. We are however looking at subsets of cell types and we need to do that to be sure we have the right subset. This may not be the case with what you are looking for it really depends on what your target cells are and what they express. This article below does several things I already do typically in making my panels and running flow.

Article A user’s guide to multicolor flow cytometry panels for compr...

Also, Purdue has an archive of all sorts of invaluable information http://www.cyto.purdue.edu/hmarchiv/index.htm

Ok so for instance I will use your flow for an example I have not done work with Platlets so I can't go into specifics, but I can use what I found in a quick google search.

Platelet Activation and Platelet–Leukocyte Aggregates in Type I Diabetes Mellitus October 2018 Clinical and Applied Thrombosis/Hemostasis 24(5):107602961880586DOI:10.1177/1076029618805861

This Paper used FSC and SSC and then CD41. CD41 shows to be high on Megakaryocytes and platelets. I would also add CD45. Since CD45 is pretty restrictive to Hematopoietic cells (except Erythrocytes and Platelets) it would be a great marker to use for specificity in conjunction with CD41. So your CD45- CD41+ cells should be "Platelets". I would pick a strong fluor like APC, PE or BV421 for both of these markers, PE on one and APC on the other for example. Yes I would always run compensation this is vital anytime you run flow and typically it always seems I have way more control tubes than sample tubes sometimes.

Do you all have a flow core in the University they should be able to guide you to these resources and help. If not... does your university got a job opening J/K...

John Powers Thank you very much for the articles and ideas you have given me, they will be very useful in the very very near future.

We do have flow core at my University, nevertheless, one must always seek knowledge from different sources :D

Glad to be able to help Aleksandar Dishkelov If you need clarification on any of them just let me know! Good luck