In my experience, it is possible to fix annexin V stained cells and see great results on the flow. Stain cells with annexin V (in calcium-rich binding buffer). Wash cells in binding buffer to remove unbound AxV. Fix in your choice of fixative (diluted in binding buffer). I use 1% w/v formaldehyde and this works fine.
The big issues:
Keep binding buffer present. As soon as you reduce calcium, the AxV simply falls off.
Washing cells can induce some changes in cells as, if they are undergoing apoptosis.
The major problem of fixing, in this case, is the permeabilization of the plasma membrane. Thus you don't will be able to use PI, wich is very important in the apoptosis analysis.
Dear laura,
Thank you very much i will look at it..
We tryed 1% PFA and see no big difference between fixed and non fixed cells...
I ll read you protocol and let you now
Thank you
Jean-Philippe
Dear laura, benjamin and Flavia...
Thank you very much i will look at it..
We tryed 1% PFA and see no big difference between fixed and non fixed cells...
I ll read you protocol and let you now
Thank you
Jean-Philippe
Dear Jean Philip,
thanks for starting the discussion and the information on fixing Annexin for flow cytometry. We once switched to a different assays to avoid this discussion. We use the fixable live-dead stains from Invitrogen to stain the necrotic cells before fixation (2% PFA). You can then store the cells short term in the refrigerator or for long term freeze them. Thaw them, permeabilise and you can then stain for apoptotic cells using an antibody to cleaved PARP (cPARP, when you work on human cells). In addition to that you can stain for proliferating cells using anti phosphoHistone3 antibodies or DAPI (depending on the cytometer you use). Since you fix the cells anyway, antibody staining is only one step further and you have information on necrotic, apoptotic and growing cells in one assay.
We published that together with Wolfgang Hartmann several times and this is the link to one of the publications.
https://www.researchgate.net/publication/23317296_Insulin-like_growth_factor-1_receptor_acts_as_a_growth_regulator_in_synovial_sarcoma or see the attachment.
Hope this helps.
Best regards
Elmar
Article Insulin-like growth factor-I receptor acts as a growth regul...
In my experience, it is possible to fix annexin V stained cells and see great results on the flow. Stain cells with annexin V (in calcium-rich binding buffer). Wash cells in binding buffer to remove unbound AxV. Fix in your choice of fixative (diluted in binding buffer). I use 1% w/v formaldehyde and this works fine.
The big issues:
Keep binding buffer present. As soon as you reduce calcium, the AxV simply falls off.
Washing cells can induce some changes in cells as, if they are undergoing apoptosis.
Thank you very much andrew!!
I'll try and let you know.
Thanx
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