In my experiment, I treated BMDC (50 000 cells/well) with different concentrations of adjuvant with or without OVA protein ( 80 ug/ml) and incubate them for 4 hours. After washing with PBS, I co-cultured them with B3Z cells (50 000 cells/well) and incubated 18 hours at 37 degree incubator. I evaluated the response with Z buffer by incubating 37 degree at 1-4 hours. OD value was measured at 595 nm. At the end of the experiment, my OD value was so low ( same as negative control). Also, SIINFEKL (1 ug/ml) OD was low (Absorbance was 0.200 at 595 nm.) What is the reason for that? Is there any missing step in my experiment?
Thanks for your help.
Hi Nilgun,
I always used OT-Is for cross presentation assays, I never tried B3Z cells but I don't see why your assay shouldn't work. After the pulse, I also always washed with medium, not PBS, but that shouldn't make any difference. In my experience, the 80ug/ml OVA may be a bit on the low side and I would try and perhaps increase the concentration. That said, SIINFEKL should have given you a whooping response. Have you tried activating the B3Zs with plate-bound aCD3 or PMA/ionomycin to see what is the maximal level of response that you can expect? Perhaps your B3Zs have lost the reporter - I used those cells for other purposes and I saw that happen... Also, as an alternative to using the b-Gal reporter, you can always check IL-2 by ELISA.
Finally, if you have access to OT-I mice, I would try and take some T-cells from the spleen and use those at least to establish if your problem is with the T-cells or with something else. You can even pre-activate the OT-Is in vitro, which gives you a memory-like population that gives much stronger response than your naive cells (read out IFNg by ELISA). I don't think this should be necessary for something as straightforward as directly added OVA, but it is an option that I can give you a protocol for if you need.
Let me know how things go.
Cheers,
Pavel
PS. As an afterthought, I hope that when you say BM-DC, you mean Flt3L-induced BM-DCs, not GM-CSF DCs because those are mostly macrophages. See Helft et al. Immunity, 2015 for details
Hi Pavel,
I will try to activate B3Z by addind PMA/ionomycin to see the max. response.
Also, I used GM-CSF induced BMDC. There are many studies for cross presentation assay by using GM-CSF induced BMDC in the literature.
Thank you for your attention and suggestions.
Hi Nilgun,
I'm aware of the amount of papers published using GM-CSF DCs, the system has existed for a long time and has been used extensively. Unfortunately, it turns out that the resulting population after the GM-CSF culture is mostly macrophage-like - an inconvenient fact that a lot of people choose to ignore (see the paper I referenced in my pevious post). Depending on what type of experiments you are doing, it might not matter that much but all I'm saying is that if you want to work with a model system that is closer to actual dendritic cells, using Flt3L is better in almost every aspect. If nothing else, I thought you might want to know that there have been some controversies about GM-CSF DCs.
Anyway, good luck with your experiments!
Cheers,
Pavel
I understand your point. I will induce BM with Flt3L and check them for cross presentation assay.
Thanks for all your support!
Sincerely yours,
Nilgün
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