I have read several papers which use Annexin V-FITC/PI with or without Hoescht DNA staining method in adherent or non-adherentcells, followed by flow cytometry and fluorescence microscope. We plan to use fluorescent plate reader, and I do understand that platereader will only record total fluorescence and may not be able to distinguish cell cycle, but I want to know if anyone has compared the methods and if so what was the difference obtained. If the sensitivity is compromised in the plate reader what is the level?
Also how best to capture floating cells, if protocols recommend aspiration. Will spinning the plate at 400g, sediment floating cells, before adding dye-binding buffer?
Hello Binal,
I think Benjamin's answer above is excellent and that flow cytometry will almost always offer you more information while plate based assays may be simpler, easier, and cheaper. In answer to the latter part of your question I have worked with non-adherent cells in a 96 well plate format and found that a 3 minute 400xG spin followed by an upside down tap on a paper towel is generally enough get rid of the wash and to keep the cells in the plate throughout the staining and washing process. I often use this technique to stain cells for large scale flow staining. Good luck to you in your experiments.
If your plan is to differentiate apoptotic cells from purely dead cells then you really need a flow cytometric or fluorescence microscopic approach. Being able to identify the fluorescence signal of individual cells is critical in this situation, since dead cells will be labeled by both PI and Annexin-V. A broad-brush, population-level assay with a plate reader probably won't provide you with enough information, although I suppose you could attempt to infer apoptosis from an increasing FITC signal in the absence of any PI fluorescence (perhaps an overall FITC/PI ratio would help here?). However, you'd struggle to distinguish between apoptotic and dead cells within a population, since both would display heightened FITC fluorescence. Basically, if you can find a flow cytometer, use that. If you can't, try a fluorescence scope and settle for a smaller "n". If you really have to use the plate reader, then think long and hard about where the fluorescence that you're measuring is actually coming from (i.e. what do the individual cells "look like" in terms of their viability"). Hope this helps, and good luck!
As everyone said, a plate reader cannot distinguish between early apoptotic and dead cells. So go for FACS if you want to measure apop and dead cells.
To collect the floating cell, just use a pipette to draw them from the well plate and put it in a sterile tube, eg. 15 ml centrifuge tube (depend on the volume of media in the well). One tube for one well. Then centrifuge.
Thank you all for your valuable suggestions. I am looking for an assay to complement Mtt result where an initial increase in proliferation is observed followed by a decrease. Is alamar blue more sensitive than MTt? Or should I look into Ldh or caspase 3? I need more than one method? Flow is a little inconvenient and want to d it only when I am as
Flow is a little inconvenient and would prefer to use it as the last option. Does any one have recommendations for Caspase3 method i.e. which companies kit is more economical and yet sensitive. How about Tunel assay? I am new to apoptosis but want to develop some techniques in the lab for long run as well.(I apologize for half-responses previously not a good typer on cell phone).
I sincerely appreciate everyone's time in writing back and scientific sharing of knowledge. Thank you.
The best way to detect apoptosis via Annexin V/PI staining is through flow cytometry. It is easy, convenient and the output is very reliable. If you want an alternative method, you can use TUNEL staining. That is also very robust and well accepted method of detecting apoptosis.
Hope this helps.
I have used a very good kit from DB Pharmigen to measure activated caspase-3 in apoptotic cells by Facs. it is PE activated Caspase-3 apoptosis kit Cat: 550914. For viability/necrosis I used a Zombie Red fixable viability kit from Biolegend (cat: 423109). there is abig choice of Zombie dye with several fluorophore produced by Biolegend...you can choose the dye according to the cytometer used.
the protocol is very simple : label first the cells with the Zombie dye according to Biolegend product data sheet protocol until step 5 ( i used PBS+1%BSA for the washing step), then switch to the activated caspase-3 kit protocol.
the cells need to be labelled first with the Zombie dye because for the PE-caspase-3 labeling the cells are permeabilized.
Much better test for apoptosis than AVfitc/ PI
good luck
I found TUNEL assay more accurate (to determine late apoptosis) than Annexin V. Roche has a very convenient (although little expensive) one step "In Situ cell death Detection Kit # 12156792910 for red and have green too) and colabel it with Nucei stains - Dapi, TOTO-3/ what ever you have. Dapi confirms the morphology, besides total cell count. Also, can detect active form of caspase 3 band by western blotting .Good antibodies for that too besides cell staining.
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