6 replies to this topic

[sabi67]

Hi,

I'm going to do several FACS experiments using between 2 and 4 antibodies. My question may appear maive but I'm wondering if I should add each antibody to each sample separately or if I can make an antibody mix  and then add the mix to each sample. Is there a risk that the antibodies bind to each other in the mix or not?
I also read a topic about not using trypsin to detach cell in culture when using Ab against membrane receptors? My targets are not receptors but are protein membrane so I guess I shouldn't use trypsin neither?
Thanks in advance for any advice  

Edited by sabi67, 11 November 2009 - 09:23 AM.

[wirly]

sabi67, on Nov 11 2009, 09:17 AM, said:

Hi,

I'm going to do several FACS experiments using between 2 and 4 antibodies. My question may appear maive but I'm wondering if I should add each antibody to each sample separately or if I can make an antibody mix  and then add the mix to each sample. Is there a risk that the antibodies bind to each other in the mix or not?
I also read a topic about not using trypsin to detach cell in culture when using Ab against membrane receptors? My targets are not receptors but are protein membrane so I guess I shouldn't use trypsin neither?
Thanks in advance for any advice  

Depends on if the antibodies are directly labeled or not.  If they are, then most likely a mixture of all will work, particularly if the antibodies are very specific MAbs.

[pesji]

For sure don't use trypsin because it will chop off the surface proteins   As for the labelling I usually do it step by step and washing each time in the meantime, but if they're not giving any background and raised in the same species why not  

[grayhair]

sabi67, on 11 November 2009 - 09:17 AM, said:

Hi,

I'm going to do several FACS experiments using between 2 and 4 antibodies. My question may appear maive but I'm wondering if I should add each antibody to each sample separately or if I can make an antibody mix  and then add the mix to each sample. Is there a risk that the antibodies bind to each other in the mix or not?
I also read a topic about not using trypsin to detach cell in culture when using Ab against membrane receptors? My targets are not receptors but are protein membrane so I guess I shouldn't use trypsin neither?
Thanks in advance for any advice  

I use premixes all the time.  Also I used trypsin on skin biopsies and have never had a problem!

[Piersgb]

I think you can only really add a mixture of antibodies if you know that they will not cross-react and that you can also detect them separately and clearly. You'd also need a pretty good flow cytometer with several different lasers/detectors to then be able to differentiate between which antibody has bound and which is fluorescing in the mixture.

I think it would be best to start adding antibodies individually so that you can say for sure that any result you are getting is due to the one antibody that you have added. I'm assuming that you are also using isotype controls?

I agree with everyone else about the tyrpsin. To protect your membrane bound proteins it would be sensible not to use trypsin. You could always try this at a later date to see how using trypsin affects your results.

[grayhair]

Use the pre-mixes

[Rsm]

You mean that you have different antibodies labelled with different colours, right? Then you anyway need to do to single stained controls, unstained, and all together. I usually put all of them together at once for the total.
Regarding Trypsin: I think it doesn't matter much if you make a brief incubation with 0.05% trypsin/EDTA (like 2-3minutes) for cell cultures. If you have primary cells (biopsies, organs) you should NOT use Trypsin. Better use Dispase/Collagenase, incubate with 3mg/ml in complete medium @37C for 30mins or longer.