When I label my PBMC with my IgG-PE or PECy7 I have a significant shift by comparison with non-labelled cells. With IgG-FITC or APC, none shit is observed. I am already working in BSA1% / Human serum 10%, and label is done after 30min on ice. Is it again aspecifity? How can I avoid this shift?Should I really avoid this shift? Thank you in advance.
Hmmm. There's a few things here:
1. When you say significant, is it really a large shift or is it simply a shift you call significant because you actually want no shift. Isotypes sometimes do give you a shift because of non-specificity, but the shift you observe with the isotype controls may in fact be small relative to the antibodies you use for your antigens. Have you done a direct comparison?
2. If the FITC and APC give you no shift, why not just use those fluorophores? Are you planning to do multicolor flow? There are plenty of other colors you could use that don't overlap spectrally. Check out this web-site:
https://www.lifetechnologies.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html
Of course you may be limited in using these dyes because the antigens you're interested in only comes with these dyes. If that's the case, see answer 1. I would also try sourcing your antibodies from different sources. It could be a supplier/batch issue.
Good luck, let me know how you get on!
Isotypes sometimes do give shifts when compared to unstained cells but what you should note that the shift is under 10^3. The best negative control is in the range of 10 to 10^2. If that's where your isotype control lies then you don't need to worry because your positive labeled cells will come in the range of 10 to the power 4 (10^4). And you can place your gate there. If your labelled cells shows peak below 10 to the power 3 then chances are that your cells didn't got labelled properly.
Normally when I label my cells I do it for 1 hr on ice. That clearly separates the isotype labeled cells from the positive cells in a Flow cytometer.
Thank you for your quick answers!
When I talked about shift it is non labelled cells: 10^2 or less (MFI less 200) and with IgG 10^3 (MFI around 600 for PE 800 for PE-Cy7). MFI / shift are higher with 10µL of antibodies than with 5µL. I can observe 2 peaks, and the one that is "positive" corresponds to monocyte population.
I need 4 colors, as I will perform multicolor antibodies. And, as much as possible, I want use antibodies we already used in the lab, for economic reasons!
I will try longer time on ice as suggested. I ordered Fc blok from BD, do you think it can help?
Daniel, what do you mean by direct comparison?
From my experience it is also worth seeing how bright your stained sample/ population is in the end as a small shift in your isotype control will be less important if you stained sample population is very bright, hence you can distingush the population you are interested in very well. Bright populations (if well set up and compensated) are somewhat easier to gate on in multicolour experiment/ dotplot.
By direct comparison I meant have you compared your isotypes directly to your antibodies. It sounds like you already have!
I concur with Kasturi and Christa - if the antibody population is much brighter than your isotype (at least 10^4 or greater) then I don't think you need worry too much.
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