Help-apoptosis of adhesion cell - apoptosis detection by annexin V binding on adhesi (Mar/27/2005 )
Hi, I want to do apoptosis assay by annexin V binding on adhesion cell lines and use flow cytometry to measure. However, the backgroup for FITC-annexin V binding on untreated cells is very high. Is there any special protocol or suitalble kit to use on adhesion cell lines?
Thanks a lot!
I am having the same problem with A549 alveolar epithelial cells. Has anyone used it with specific cell line and any tips on trypsin vs edta or commercial preps for harvesting the cells?
I used to do Annexin V staining for FACS on adherent cell lines. I found that my trypsinisation using commercial mix of trypsin/EDTA only resulted in a very small percentage of damaged cells <1%. I guess that it is vastly dependant on the cell type.
Have you considered staining the cells on cover slips and examining by IF and just doing cell counts?? Or fixing cells prior to harvesting, not sure if either would work but could try. The other possiblity is that there is any number of commercial kits these days for assaying apoptosis you could check these out.
Sorry I haven't been much help.
I have used the BD Pharmingen Annexin V_FITC detection kit extensively. It provides AnnexinV-FITC as well as propidium iodide so you can determine cells that have an intact membrane. This allows detection of early apoptotic cells.
If you are getting high background you may need to add a blocking agent such as 1% BSA or IgG to your buffer. Also, using propidium iodide you can tell if your cells are being damaged from your treatment (removal from the flask).
The BSA you mentioned.. do you know what i does with the cells or the sample to reduce the background?