Dear All,
As all we know that ETBR and PI are used in differentiating early apoptotic cells from late apoptotic cells in combination with Acridine orange (AO) and AnnexinV-FITC respectively. Combinations are popularly known as AO/ETBR and AnnexinFITC-PI staining. However, AO/ETBR seem less frequent than Annexin –PI (or 7-AAD). However, I have not passed through protocols or kits that uses Annexin with ETBR or AO with PI (or 7-AAD). Does this pose a real issue in use of ETBR with Annexin or is there just some sort of convention or it’s just because of my lack of information? Thanks.
Not sure about interactions of various dyes with annexin staining (other than needing to make sure that you have enough spectral separation to avoid channel cross-talk), but I would actually recommend something totally different instead of ethidium bromide or propidium iodide if you aren't constrained by what reagents you already have: Invitrogen SYTOX Green, Invitrogen SYTOX Orange, Biotium EvaGreen, Biotium GelGreen, and Promega CellTox Green (expensive) seem to work the best in terms of giving clear staining and staying out of nuclei of live cells during prolonged incubation, as well as giving decent results with a Perkin-Elmer EnVision multimode reader looking for fluorescence of permeabilized cells. Several other dyes (with a greater range of colors) share these characteristics except for not working well with the plate reader (despite much finagling with filters and other reader settings). Most of the dyes work fine for fluorescence microscopy. See Article Evaluation of Impermeant, DNA-Binding Dye Fluorescence as a ...
I would avoid the use of AO especially for microscopy as it is not particularly photo-stable. Additionally at low pH or high concentrations the dye stacks and you get a meta-chomatic shift to red fluorescence emission. Many years ago many labs shifted to using PI as a fluorescence exclusion dye as it is much less toxic than EB. Latterly SYTOX dye are reported to be less toxic than PI. You can get annexin-V congugated to many different fluorophores so have a wide choice to suit your equipment. Depending on your equipment and budget. SYTOX green in combination with Annexin-V Alexafluor-647 would be a good combination. A word of warning, do not freeze the Annexin-V. A college of mine some 20 odd years ago went to the trouble of purifying and FITC labelling annexin-V (before it was readily available commercially) and it worked brilliantly before someone put the stock into a -20 freezer.
I have 3 different treatment groups and the control group. Could you please help as I need to perform the acridine orange assay. Do you have an easy protocol. I do this experiment in a 24-well plate.
Thanks!
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