Dear Rob, Becton Dickinson has validated kits for intracellular IFNgamma but you need to seach for IFNalfa and beta. Best would be to contact them and find out about the difficulties. Best of all, Gulderen

Hi Rob,

did you come across some well-working ABs for IFN-a/IFN-b?

Cheers,

Claudia

Claudia,

Unfortunately, no. I tried the IFNb-FITC (PBL interferonsource, 22400-3) with pretty weak results. It's an old tube, so that may be part of the problem, but I only saw a mild shift in fluorescence, not enough to really hang my hat on. We get decent intracellular staining for IL-10 and TNFalpha, but I really with there was a better reagent for the type 1 IFNs. Let me know if you find anything better!

cheers,

Rob

I have the exact same problem with the IFNb-FITC (PBL interferonsource, 22400-3) as you Robert. When I stimulate BMDCs with L. acidophilus NCFM that always induces a high IFNb response in these cells I only get a very weak shift in fluorescence. However, mine expired a year ago... Let us know if you buy a new one and try it out!

Dear all,

Just found your chat on that particulat AB from PBL. I just ordered and want to try it in ICS/ FACS. Is there meanwhile any clue how to improve the assay?

I will go for different TLR stiumlations in PBMCs and try the PBL one as well as one from Affymetrix, which is so far not vaildated for Flow.

I will be pleased if I get some hints!

Best Susanne

Hi Susanne,

I haven't tried this in a couple years (moving institutions) but am probably going to revisit it as we're getting a new flow cytometer that is supposed to be a bit more sensitive.  I will add one thing to the discussion that may be helpful: many types of cells that express type 1 IFN do not actually express huge amounts.  Specifically, many macrophages only upregulate IRN-beta mRNA 5-10 fold with most TLR stims, considerably weaker induction than you would get for TNF-alpha, RANTES, IL-6, etc.  So I suspect that the low IFN-beta signal I see by flow actually reflects the real biology and not just a technical or sensing problem.  Plasmacytoid DCs certainly express way more IFN-beta and IFN-alpha and these might be a useful positive control -- I've just never had time to test that out.  

cheers,

Rob Hagan

Using Golgi blockers for inhibiting protein secretion will increase signal in your assays. Good luck. 

Dear all,

I have redone my assay with a brand-new AB (PBL) and of performed golgi block. While other cytokines (TNFa, IL6 etc) work perfectly fine, I cant get a convincingly positive signal for IFNbeta for either in vivo or in vitro assays. Any suggestions still welcome!

Hi Claudia,  

Have you considered adding the golgi blocker later? I would induce and  incubate more time but adding the golgi blocker at the end. Secreted Interferon induces more interferon expression, in a paracrine effect, so I would add the blocker later (eg: in the last 8-12 hours of a 48-72 hrs stimulation....the second wave of induction is stronger..good luck!!

Hi guys,

is there an update on your experiments with measuring IFNB by FACS?

Best,

Falko

Falko,

I haven't re-tried intracellular clow staining for IFN-beta or IFN-alpha in a while.  I still wish there were better reagents, but I certainly think the low signal is tied to the biology.  By multiple readouts (RT-PCR, ELISA) we see that the fold induction of type I IFNs from a lot of cell types is low compared to other genes like CXCL10, IFIT1, Mx1, etc.  If there are going to be better flow methods for type 1 IFNs I think that either someone will have to work really hard to make a much better antibody (unlikely) or use a reporter mouse (like Richard Locksley's -- I think it's IFN-beta-EYFP). 

Rob

Hi!

We have been using IFN-α[2b] Alexa Fluor® 647 Mouse anti-Human   Clone  7N4-1 (RUO) from BD. After overnight stimulation of human PBMCs with TLR7/8 ligand together with BFA. Can't tell you what cells are producing it since it was only a positive control though, most likely pDCS. Haven't tried beta-interferon-

Best