Dear Priscila,

I think both PE- and APC-conjugated antibody work very well, even there is some compensate existing, we have no other choice, just try to adjust it, the data is still reliable.

Genemedi got a rich experience in virus production with many fluorescence labeling, you could find more information on https://www.genemedi.net/i/lentivirus-packaging

Hope this may help you.

Hi Priscila

I guess it depends on the lasers you have available (and what filters you have).

I'm surprised you're having trouble with the PE/GFP combination. We routinely do this combination: DAPI, APC/Cy7, PE, AF647 and GFP. With this staining (and a little compensation) we can resolve all our 7 populations of interest that we study.

As for your work, perhaps you might want to try AF647 - it's bright and commonly available.

Good luck.

Al

I'm using a CAlibur FACS machine and my problem was: I used PE-conjugated mouse IgG isotype control in one tube (it wasn't supposed to stained anything) and in another tube I used the specific antibody (PE conjugated too). Both of then showed the same results. I mean, both Ab isotype control and specific have detected the same thing. I think that the GFP fluorescence are masking the true result.

In FACS Calibur I routinely use APC ou PECy5 when I want to compensate with GFP. It works very well.

If you are using a facs calibur I would use APC as your second colour that way you wont get any spill over into your FL-1 where you are detecting you GFP. Plus it is nice and bright.

PerCP: Can be excited with the 488 laser and emits in FL3

I've never had problems compensating between GFP and PE using the Calibur. What you describe could be a problem with bleedthrough. Check the machine filters with your FACS expert. However correct controls should allow you to compensate properly.

You can do a test by staining the same samples with APC or PECy5 as mentioned above. And see if you get the same results.

If isotype and proper antibody look the same, it means that probably your isotype control binds non-specific or your proper antibody is not working. The best for GFP is to use FL3, it is much easier to compensate the stainings. However, if the staining is not specific there is little chance to compensate it.

I was about to post my response and saw that Alasdair Russell had pipped me to the post - Alexa Fluor 647 is a great readily available and easy to conjugate probe with a good quantum yeild - I've had great results with this probe as a co-stain for many years now (ex/em = 650/665nm) . Although mole for mole something like CY5 is brighter, this probe resists photobleaching brilliantly - highly recommended!

I've checked the antibodies and I'm sure both of them are ok. When I stained transfected 293T cells everything works so good, I mean, PE-IgG control and PE-specific Ab work as I espected. But when I stained lentivirus transduced 293T cells I had that trouble (IgG and specific Ab staining equally). The lentivirus vector has GFP gene as reporter. I am thinking if lentivirus could disturb the staining (some viral protein)... I don't know

Hi, when GFP is well expressed it is very bright and emits into PE channel (FL2) as well as in PECy5 channel (FL3). The signal you see in PE channel in your case could be mainly GFP signal. especially if the antigen you stained for is not well expressed by your cells. You need to correct PE signal (using cells labelled with GFP only-NO staining for PE) by removing GFP signal from it. That's what we call compensation. However, If your PE signal is dim you will not be able to see it in bright GFP cells even after compensation. In this case you need to choose a different colour emitting far away from GFP or excited by a different laser like AF647 or APC if you have a red laser on your FACSCalibur (FL4 channel).

I think in this case it's not a question of antibody specificity. If you can't separate PE signal from GFP one, you can't tell if your PE-conjugated primary antibody is working or not.

If GFP emits into FL3 too, there is a problem if I use PerCP? I don't have APC right now.

GFP emits less light into FL3 compared to FL2. You can use PerCP but remember to compensate between FL1 and FL3 (you can do it via FL2 compensation matrix). Always have single colour controls when you are dealing with more than one colour. It should work.

APC is the best option, when you have FITC, GFP, AF488 stained samples. I always use APC if its FACS calibur or PB on LSR II. Usually, if you only have these two colours then sometimes one does not even need to do compensation. However, it is recommended to compensate because cell types are different, some autofluroesce and some not. What are you using for compensation? your cells or beads from BD. I recommend using beads. Also, having Fluorescence minus one (FMO) ctrls will help, also suggested by Ayad.

Im using my own cells line to compensate the FACS. I will test the staining with PerCP and I hope everything goes ok.

PerCP its ok but first compensate automaticaly and when stay in course or after take a resul in the flow software re-compesate manauly becuase some time moved ligthtly.

And depens of time money and stetic that u need on yors dot plot or final results soemtimes is good Alex-647 and 488 makegood contrast and have very low traslapation (or check others because its dependent of your cytometer and FLs that have)

What Cytometer are you working on? I would recommend PerCP-Cy5.5 instead of PerCP, the quantum yield is much higher as is the fluorescent stain index. PerCP has a stokes shift that is too large so a lot of the energy is lost before light emission. The tandem will give you much brighter signals and can be used on any flow cytometer with a far red emission filter (so e.g. FL3 on the Calibur)

Best,

Felix

I do not understand why you cannot compensate. I have used GFP expressing cells with PE and gotten very clean compensation on a FACSCalibur. Recently I have tried the same with 7AAD and GFP expressing cells on LSR II and gotten clean color separation. You may need to spend time with this. Begin by using single colors and finally combine them and fine tune the separation.

Good luck.

Hi Priscila,

I think you can separate GFP and PE signals. Before you start analysis, you had better make compensation with GFP positive PE negative cells and PE positive GFP negative cells. If you use double positive cells for adjusting compensation, it is very hard.

Good luck,

Yoshio

Hi, Priscilla, join to others who never had any problem with measuring GFP together with PE. Actually, in my panel and using FACS Canto II, I had GFP (on the FITC channel), PE F4/80, PerCP CD45.2, PE-Cy7 Gr1, APC CD11b and APC-Cy7 CD11c. For compensation instead of GFP expressing cells, as they were not many (appr 10%) I used FITC GR1 stained cells. Ok, in another panel - GFP (again, on the FITC channel), PE-Cy7 CD4, PE CD25, PerCP-Cy5.5 CD3; compensated on the same cells (splenocytes, GFP under FoxP3 promoter). Both cases caused no problems, populations were clear and nice. If you feel not secure with compensation - use FITC labelled cells for compensation. Good luck!

FITC and GFP have no where near the same spill-over to PE. Using FITC for GFP compensation is just wrong, sorry to say this! I use GFP commonly in my flow assays and the spill over varies quite dramatically (I have to look up my 'normal' settings on the Canto II but it's definitively a few percent difference, so this has quite some impact). In theory, GFP and PE should not cause too much of a compensation problem if you use good single stain or FMO controls to validate your PE (FMO becoming very important if you deal with weak PE signals and multi-colour stainings). I agree with the other posts that propose using a far red dye on the red laser (APC, Al647), if you only have two colours to analyze this will give the clearest results since there will be no significant spill-over whatsoever. What exactly are you staining for?

Felix, I am aware that it is not recommended to compensate GFP with FITC but sometimes you have no choice at all - in that case when I had to do this I expected 0-5 % 0f GFP+ cells among all other cells (without any possibility to discover whether its there or not before measuring). As I saw clearly distinct population of GFP+ cells in some samples and none - in others, I assume that it worked.

Alexandra, But the way the problem is put, it appears that all cells are expressing GFP. That way it should be very easy to see these cells in the FL1 (FITC channel), First, what should be done is to set these cells clearly separated in this channel, then stain PE on the same cell type (non-GFP expressing). Afterwards, stain the GFP expressing cells with PE and fine tune the color separation continuing with settings from the previous compensations. It works for me.

Regards

Alexandra, are you using a digital flow cytometer (e.g. Canto, LSR, Fortessa or similar machine from other company than BD?). If you have to compensate only one colour this is fairly easy if you use a GFP/PE dot plot and switch to a biexponential display, you would see correct compensation compared to over-/undercompensation directly. If all of your cells would express GFP, I would recommend to spike in a GFP- population with similar autofluorescence (normally a non-transfected cell or a culture cell of the same tissue origin) so that you can control what's GFP and what is not, but even if you have all of your cells expressing GFP you should see the PE signal coming down to 0% spill-over below 20% compensation (normally far(!) less, except your put your PMTs in a very odd range). Keep in mind that autofluorescence is never compensatable, so therefore even if 99.9% of your cells would express GFP and you know your cytometer and fluorochrome a little you should be able to compensate this even without a negative population at hand. I agree though that it is possible to use the FITC as a rough approach, I normally use my 8-colour comp also if I switch from FITC to GFP (works for any other dye exchange also....) and do a little adjustment for the spill-overs to put the cells into the right place. Just keep in mind that this will never be 100% accurate.

Hi, Felix :) I was using Canto II that time and, unfortunately, I ought to use 6 colors to fish out population of cells which potentially could potentially express GFP upon activation (up-taking tumor material and processing it and thereafter become activated). As I was looking for those cells in the tumor tissue and surrounding "healthy" tissue I was not guaranteed that I would have many of them. In fact, they never were more than 5%. Therefore, knowing from the previous experience how do these cells look like in the tumor cell suspension upon using applied panel I risked to use FITC for compensation. It worked. I had no GFP positive control. Again, you are talking about GFP-enriched cell suspension, where almost all cells express GFP - but which choice do you have that almost all cells do not (and must not) express it?

I would suggest a gfp reporter mouse if you can get hands on them. For compensation also gfp transfected cells would work, remember that you will always compensate the fluorochrome, not the antigen. As long as your pmt settings stay the same, you can use ANY gfp+ cell for an accurate compensation. If there is none around in your department, though, adjusting the fitc compensation still may be your best option. As you said, as long as you know your cells you can readjust the comp easily on the fly.....

that's how I ended up with FITC :) anyway, thank you, Felix, for interesting discussion!

I have done a FACS with GFP cell (about 5% if I gate on CD4), and have compensated with FITC, along with PB, PE, Percp and APC. WIth that said though PE is probably the worst colour to choose because I think it is right next to the GFP channel. I sometime get compensation up to 25%.

Try APC and PB, I don't think you need to compensate at all with these 2 colours.

Hello,

You may use something in the far-red spectrum such as PE-Cy7 instead of PE. you won't need to compensate. but check the laser configuration of your cytometer if you have a far-red laser

What cytometer are you using? Some thing on your second or third laser would be a good option or else APC 

You got many good answers already. But heres my comment in case you are still working on your problem.

PE and GFP together can be difficult, particularly if you do not have automatic compensation routine in your software, I used to use an old version of CellQuest, so manual compensation only. There it was very challenging, but still it can be done. For setting up, try GFP-transfected cells and borrow an antibody to a well-expressed pan-leukocyte antigen (Such as CD45) I used to have a fairly ok setting for GFP, PI and PE on a FACScan I (CellQuest). But I had help from a very patient and experienced facility manager. Now I am at a different place (without such a facility manager) where we have a FACScanto running FACSdiva (automatic compensation , works great, otherwise the most unintuitive and convoluted piece of software I know of...) Plus make sure you are using GFP and not (by mistake or unknowingly) YFP (still looks rather green to the eye), then it is literally impossible to separate from PE.

As others have pointed out above, to make life easier switch the fluorophore to APC of PE-Cy7. For us that worked great. But before ordering first check your available filters and laser(s) using this link (also pointed out before, by Juan Sebastian Pappalardo):

http://www.bdbiosciences.com/eu/s/spectrumviewer

Good luck. It can be done! Do not lose hope!

Thomas :-)

Dear Priscila,

I think both PE- and APC-conjugated antibody work very well, even there is some compensate existing, we have no other choice, just try to adjust it, the data is still reliable.

Genemedi got a rich experience in virus production with many fluorescence labeling, you could find more information on https://www.genemedi.net/i/lentivirus-packaging

Hope this may help you.