What are the factors that can result dead cells in flow cytometry analysis of murine tissues?
Hi Iqra
As Okan already asked, we really need more details to answer this question well.
There are many factors that can induce cell apoptosis before flow cytometry.
You mention you are working with murine tissue. Typically you dont analyse "tissues" with flow cytometry. Blood counts as a tissue, but normally you isolate cells from blood, and then analyse the cells.
What type of cells are you isolating? Some cell types are more fragile than others.
If you are using frozen cells that are thawed, a bad freezing and thawing method will cause a lot of cells to die between thawing and running them through flow cytometry.
If you are using cultured adherent cells, a bad cell disociation method can cause cells to die (like too high concentration of trypsin).
Overall we need more specific information from you on what you are doing.
How did you isolate the B/T cells from the tissue? This step can cause a lot of cell death if not done properly. What is the wash solution, what is centrifugation speed, etc. I would need more details.
If you are looking at B and T cells, i assume you are immunophenotyping/ labelling with antibodies? How do you label the cells? Is it at room temperature or 4C? Is there sodium azide in the antibody staining reagent? How long is the staining done?
Last, it sounds like you are determining that a lot of cells have died because your T cell number % is very low? To be sure you need to incubate your cells with live/dead dye; ratio of cell phenotypes is not considered a marker for cell death. Some common live/dead dyes include 7AAD, TOPRO, and PI.
Again, it is hard to answer your question. You give us very little information. There are so many steps in flow cytometry that if dont improperly can contribute to cell death. Please mention how you isolate your cells, how you do your staining, etc.
After dissecting mice I put organs in PBS clean them and kept in RPMI. I prepared mononuclear cells.
For spleen I used red cells lysis buffer. Placed spleen in buffer for 4 min then centrifuged at 2400 RPM for 2min discarded supernatant. Added 600ul PBS, centrifuged at 2400 RPM for 2min removed supernatant. Re added 200ul PBS, centrifuged at 2400 RPM for 2min. Used 1ul both B and T cells antibodies CD4, CD3, CD8,IgM,IgD,CD19 , cover with a foil and placed them at 4c for 30 min. After 30min centrifuged them for 2min at 2400 RPM, remove supernatant added 600ul PBS , centrifuged followed by addition of 200ul PBS. Discarded supernatant and filtered them into tubes.
For lymph nodes I followed the same procedure except the use of red blood cells lysis buffer. I did all the steps by keeping samples on ice. I started dissecting 20 mice in the morning around 7 and finished flow cytometry at 1 pm. I hope I answered properly.
Unfortunately I dont work with mice cells, instead with human and cow, but should be close enough.
A good flow cytometry experiment is like building a house, you need solid foundations. It takes a lot of time and it rarely works if you try everything at once as a beginner. You really have to go step by step. For example, you should test each antibody one by one.
With the information you have given me, it might not be a cell death issue, but something else. You are using multiple colors (CD4,CD3, etc...). A low % of one color marker might mean a bad antibody clone. A clone is a "version" of a antibody for the same target, and using a different clone can result in very different % even for same target. For example in human cells, CD20 (B-cell marker) clone 2H7 does not work well under certain conditions, giving low %, but clone L26 works just fine.
It could be due to bad compensation settings. If you have mutiple colors in one sample , you have to correct for each color affecting the other.... Another example is how many events are you using to get the information on %, typically 10'000 live, single, and steady flow cells are needed to be sure. A typical gating strategy is seperate cells from debris by SSC vs FSC, then get single cells by SSC-A vs SSC-H, the live cells with viability dye histogram, then get steady flow by time vs SSC-A, and only then gate with your CD/Ig markers. If you have a lot less than 10'000 cells, this can give strange results as well.
Has someone used these markers before you, with the same cell types, and validated them, done antibody titration? Has someone shown you proper gating strategies? There are so many things that could give you a low %. Do you use Fc blocking reagent? A lot of immune cells, like monocytes, will "grab" antibodies meant for other cells, and if you have very little antibodies (at or near saturation) for your other cells, this can also lower %. Fc block reagent stops this (im not sure if its same with mice cells).
Regarding your isolation, i have a few tips. Adding of DNAse I to your medias will reduce cell death. Dont vortex samples too hard to mix; better tap pellets or gently pipette. But overall it looks ok.
My final recommendation is to try, with your next two mice, to do everything the same, except do not add antibody markers, just keep cells in the same media as you would have with the markers, at 4C for 30min, to get unstained control cells. Then at the end of this, incubate cells for 15-30 minutes in a viability dye (live/dead dye, like i mentioned before, 7AAD, TOPRO, etc), then analyse on flow cytometer for that dye. This way you will know if it is problem with your process, or a problem with your antibodies. There are many protocols for live/dead, viability analysis on flow cytometry, good luck!
I would recommend talking to nearest flow cytometry expert, to look at data and protocols; it can be a complex method with many many variables that affect outcomes. It will be really hard to get a fix over the interent via text.
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