Yes, it works without problem both for direct and indirect labelling reactions. Thing you should do is to bring enough culture cells - not one tube! - to put set bars for control, then you can see FITC (488 nm ex. - 520-530 nm abs.) and not autofluorescence. You might need to move these bars to 515-525nm, for example. And antibody should be alive also...

Do you have auto fluorescence set with unstained cells on flow

Are you exposing your stained cells to light or has your antibody been exposed to light prior to staining

Are you using saturating amounts of antibody

Yes FITC is a dim fluorophore but it should work, what is the antigen you are trying to detect - is it a very low expressing surface antigen - if so you might want to shift to PE

As Elena said it works great. If you have strong expression of your gene on the surface. Also in addition to unstained cells use an isotype matched control conjugated to FITC

at the first thanks a lot for your kindly respond...

we have two problem:

1) actually when we treat MCF-7 with CD44-PE the test showes this marker is positive but when we treat them with CD44-FITC the test result turn to negative

2) i wanna detect EpCAM on MCF-7, when i detect them as single cell the percent of flow was 100% its mean the expression of this marker is great

but when this cells isolate from heterogenous population the unacceptable percent was 5 % so i dont know this low percent is resulted from the isolation technique or related to the FITC !

From your description it sounds as the problem is in the harvesting of the cells. Is the clone for the pe and fitc abs identicle ? Do you see such problem in non-adherent cella ?

Dear Pichas

i have never checked this experiment in non-adherent cella...

i wonder if you describe more about problem that you said about cell harvesting?

what is your mean exactly?

actually we treated MCF-7 in two different way:

1) we stained MCF-7 with CD44-PE and CD24-FITC from different company in one tube

2) we stained MCF-7 with CD44-FITC and CD24-PE form same company in one tube

in the first experiment approx 90% cells population were CD44+ CD24-

but in the second experiment CD44- CD24+

its seemed in two experiment just PE + and FITC - while we have same cell lin in two experiment and we expect MCF-7 CD44+ CD24+(approx 70-80%)

what is your idea about this problem!?

what the wrong is?

so some ligands are very sensitive to Trypsin, maybe you should use EDTA or scraping

second problem that might be is the compensation, try single ligand staining (only cd44 or only cd24) with no compensation then adjust the compensation from fl1-fl2 and fl2-fl1

Good luck

also please see this double staining of mcf 7 for cd44 cd24

http://www.nature.com/bjc/journal/v102/n1/full/6605468a.html

In our experience, CD24 shows a fairly low expression on MCF-7 cells and can be difficult to detect. In addition, some antibodies directed against CD24 do not recognize the core protein but associated carbohydrate structures. Thus, selection of the right antibody is critical. Which antibody clones did you use? We have good experience with SWA11.

MCF7 in general is CD44 low and CD24 high. This has been established in literature: Gene expression data and protein expression data. Do you have appropriate antibody controls to see if the CD44-PE epitope is not binding non-specifically to cells. Also, are you sure you have the right CD44 epitope since thre are CD44 variants and antibodies raised against these variants. I have done flow cytometry with MCF7 and MCF7 that has overexpressed CD44 with BD antibodies; FITC-labeled. I have almost nil expression with the parent and close to 90% with the overexpressed line. I also use EDTA for detaching cells from the palte and then spin them down low speed and perform all my reactions on ice and in the dark.

I would endorse the following points:

detach with EDTA only

Perform isotype controls for immunofluorescence levels

Perform single staining to sort out compensation

Check the clone of each antibody

Additionally, why not use a different fluorophore if you think it's the problem? There are many fluorophores available for these common molecules.

Nice discussion.

Dear Fatemeh, if according to people different Abs have different affinity to the same MCF7 culture, it means they are made for several epitops of CD24 and CD44 antigens. As we know, MAbs and Abs are the result of animal's response for administred immunogen. So, if you inject mice/rabbits with 30 aa peptide the immune response/antibody which is produced in hybridoma afterwards will have nothing in common with cell surface glycosylated receptor. And still it is an antibody for CD24/CD44 protein, only it won't recognise cell receptor but will recognise its protein in WB and IP (ELISA) when protein is dissembled by, for example, SDS or mercaptoethanole. And contrarywise, if animals were immunised with tissue (breast tumor, cell culture) lysate, MCF7 cells will be fairly recognized in flow cytometry/microscopy experiments.

In many cases this info about MAbs is even not disclosed by producer. But by request they should deliver this info, it is impossible they don't know what they injected for immunization. FITC or any other labelling you choose from the whole several hundred available from catalogue. 

I think everything is OK with your controls by now.

And one more thing, cellular biologist should have normal background education in biochemistry, basics of molecular biology and animals assays - all with handling basic methods - to achieve any positive results in the field and less "miracles" in sending out publications.

Greetings,

E.F.

Instead of using FITC, have you thought about using Alexa Fluor 488 conjugated antibody? You can use CD24 or CD44 conjugated to Alexa Fluor 488 and have the second antibody conjugated to Alexa Fluor 647 or 700. This will also minimize spectral overlap issues that you encounter between FITC/Alexa Fluor 488 with PE (a very bright fluorochrome). As with your existing experimental setup, you should include all the single and fluorescence minus one controls.

I have analyzed EpCAM expression on MCF-7 and MDAMB231 cells and they worked fine. However, I tracked EpCAM with a secondary antibody conjugated with Alexa Fluor 647. In the MDAMB231 cells, I used this secondary specifically because I was tracking the effect on EpCAM expression due to transfection with a GFP-tagged protein.