Good evening.
I am wondering if it is possible to do an Annexin V FACS staining on B cells extracted from the bursa together with staining with an antibody against surface IgM. I read that B cells do not pass the first selection step if the signal cascade fails, not necessarily because of lack of the actual surface IgM. So there should be a population of IgM bearing cells that go through apoptosis (well, there should be anyway if the receptor is strongly self reactive). Problem is, apoptotic cells go through a outer-inner membrane switch. This is what the Annexin staining relies on. Does that mean that the IgM is now "pointing" cytoplasmatically? Is it possible to do the Annexin stain and then use a fixative/detergent to permeablize the cells for the anti IgM to go in?
Hello Ori,
There is no need of doing permeabiliztion as that will only increase cell death and you may not get a high false positive picture of actual cell death.
Antibodies are taken up by live cells if you will incubate the antibody with cells at 37 in incubator. After optimum incubation, you can process your cells for typical Annexin-PI staining as IgM antibody will be already taken up by the cells. You can try the same and I hope that you get results as per your expectation.
Now you can gate your population of interest and proceed accordingly but you have to take care that emission of your IgM is not falling within FITC/PI range, if you are also using PI. This will lead to spillover. If you want to use only Annexin and your IgM is labelled with some other fluorochrome then it will work fine.
Hope it helps !
Hello Ori,
Have you attempted the stain yet? permeability of the membrane would cause cell membrane disruption and will initiate non-specific binding of annexin V to PS on the inner surface of the cell membrane. Thereby you will have false positives.
I have attempted to stain cell populations of a model organism with antibodies and Annexin V. However each attempt was unsuccessful due to massive cell death. I have attempted
1. Dual staining of antibody + Annexin V (with and without 7AAD) at the same time
2. Stain with external antibody first and then Annexin V (with and without 7AAD)
3. Internal DNA markers such as Draq5 together with Annexin V (with and without 7AAD)
Note - I stain antibodies or internal markers in ice for 30 minutes in dark, and annexin V at RT for 15 minutes in dark. Single stains are successful with FACS and FM, but not when combined.
Each time the cell death count was massive.
I have attempted to sort my antibody positive cells and then stain the sorted cells with annexin V, PFA fix and detect through fluorescence microscopy (FM), but the results are inconclusive.
The only success I got was using antibody sorted cells stained with caspase 3 and visualized under FM.
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