Hi Daniel,

1) Are bone marrow derived DC better to be pulsed than splenic DC?

bmDC are longer to obtain (6 days) than splenic DC but easier to maintened in culture than splenic DC. However if you just want to activate OT-2 cells with peptide- loaded DCs, splenic DCs are a good and easy tool.

2) How long do I need to incubate DC with the ova peptide before washing DC (to take away non internalized ova peptides) and do the proliferation assay with OT-II T cells?

Only 30 min at 37°C are required. (DC at 10.10e6/mL, OVA peptide at 5-10µM in complete medium).The time for proliferation assay depend of the technic (4-5 days for CFSE stained OT-II, 48-72h for Cr51 assay)

3) Do I need to stimulate DC with LPS (for instance), after incubation with peptide to increase antigen presentation? If yes, for how long to do before washing DCs and do proliferation assay with OT-II T cells?

Classicaly, DCs are matured with LPS 16h-24h BEFORE incubation with peptide.

If you want more complete protocoles:

http://www.ncbi.nlm.nih.gov.gate2.inist.fr/pubmed/23852701

http://www.ncbi.nlm.nih.gov.gate2.inist.fr/pubmed/23065740

Good luck!

Hi,

The answers to your questions depend on what is it that you want to look for.

As far as your other questions:

1)Both BMDC and splenic DC will work fine.

2) There is no need to wash the remaining OVA peptide (unless you want to time the response) since the peptide only activates cells in the context of MHC

3) Stimulation with LPS while using specific peptides is generally not necessary. You can get proliferation of OT-II cells using B-cells or macropages as APCs since antigen presentation (while using peptides) is not crucial.

However, if you plan to use (at some point) full OVA, then processing becomes important and then you would have to consider using LPS (once again depending on what it is that you are looking for in the first place)

Most BMDC protocols with GM-CSF work fine. The use of non-treated plastic petri-dishes for differentiation dramatically increases the recovery of BMDC at the end of the differentiation protocol.

You might want to check this other thread: https://www.researchgate.net/post/Does_anyone_have_a_protocol_for_generating_dendritic_cells_from_mouse_bone_marrow_using_GM-CSF

Hi Daniel,

1) Are bone marrow derived DC better to be pulsed than splenic DC?

bmDC are longer to obtain (6 days) than splenic DC but easier to maintened in culture than splenic DC. However if you just want to activate OT-2 cells with peptide- loaded DCs, splenic DCs are a good and easy tool.

2) How long do I need to incubate DC with the ova peptide before washing DC (to take away non internalized ova peptides) and do the proliferation assay with OT-II T cells?

Only 30 min at 37°C are required. (DC at 10.10e6/mL, OVA peptide at 5-10µM in complete medium).The time for proliferation assay depend of the technic (4-5 days for CFSE stained OT-II, 48-72h for Cr51 assay)

3) Do I need to stimulate DC with LPS (for instance), after incubation with peptide to increase antigen presentation? If yes, for how long to do before washing DCs and do proliferation assay with OT-II T cells?

Classicaly, DCs are matured with LPS 16h-24h BEFORE incubation with peptide.

If you want more complete protocoles:

http://www.ncbi.nlm.nih.gov.gate2.inist.fr/pubmed/23852701

http://www.ncbi.nlm.nih.gov.gate2.inist.fr/pubmed/23065740

Good luck!

Hi Daniel,

I have some experience with this pulsing DC isolated from the lung draining lymph-node, but I believe the protocol is essentially the same with DC isolated from any tissue. It is mainly a question of which subset (or tissue source) of DC you want to study. You should incubate the DC with the OVA peptide for 1 hr at 37oC. You don't need to stimulate the DC with LPS, and this will undoubtedly skew the immune response, so you might want to consider whether that is what you want.

I've copied some of the key information below. Full details are in the paper Hardy et al., 2012, J Immunol 188:1431.

DC stimulation of naive T cells

LNs from BALB/c (H-2d) mice were digested as described (Hardy et al., 2012, J Immunol 188:1431) and non-DCs removed by incubating with predetermined concentrations of Abs [anti-CD3 (KT3), anti-Thy1 (T24/31.7), anti-CD19 (ID3), anti-erythrocyte (TER-119)] and removal of Ab-binding cells as described (Belz et al., 2004 PNAS 101:8670). DCs were gated to exclude doublets and dead cells (propidium iodide) and sorted on the basis of CD8a and CD11b expression (Belz et al., 2007, Nat. Immunol. 8:1060). CD4+ DO11.10 T cells (5 x 10^4) were cocultured with sorted DCs (range 6 x 10^3 to 12 x 10^3) in duplicate as described (Belz et al., 2004 PNAS 101:8670). For the peptide pulsing experiments, DCs (12 x 10^3 to 24 x 10^3) were isolated from naive mice by enzymatic digestion and sorting as above, pulsed with OVA MHCII-restricted peptide OVA323–339 (25 ug/ml) for 1 h at 37°C, washed, and cocultured with 5 x 10^4 purified CFSE-labeled OVA-specific CD4+ T cells. Cultures were analyzed for proliferation at 60 h by staining with CD4–PE and propidium iodide and gating on CD4+ propidium iodide–cells.

Dear colleagues, thanks for the help. I will try the suggestions and i will report here the results.

Hi Daniel!

Might be a bit too late, but if you are still setting up your assay, let me share you that my protocol is quite similar to the suggestions you've received above, just some particular details:

- I use 96-well U bottom plates

-10,000 BMDCs + 50,000 T-cells (purified CD4+CD25, using Stemcell system and miltenyil magnetic beads), in a final volume of 200 µL

-The OVA peptide is in the co-culture the whole time (5 days). It's a good idea to take a look on your culture at day 3...if by some reason your cells are proliferating too much, if you use medium with phenol red you would observe that it turns yellow-ish, meaning that you have to replace *very* carefully (you will see a small cell "pellet" at the bottom) about half of the medium with fresh one.

(I assume, you would be using CFSE labelled T-cells, right?)

As for pre-incubating the cells with LPS or not, in my hands it is not necessary for the experiments I do; if you detect too little proliferation, you could consider to pre-incubate the BMDC for 4h with LPS (0.1µg/ml-1µg/mL is a good working range), wash a couple of times, and add to your co-culture. Beware that in this context, your T-cells would be receiving not only signals 1 and 2 (the antigen presented, and contact with costimulatory molecules), but also the cytokines produced by the BMDC as result of activation. Please let us know in the end what worked for you! good luck!

So I am doing a similar experiment but I am looking all over the web and cannot find the answer: how much OVA (whole protein) does it take to activate CD4+ in vitro?? The answer from http://www.malariajournal.com/content/7/1/88 seems to be mg/ml level. Anyone know for sure?

I read else where to activate CD8+ (cross presentation) with OVA protein takes 2 mg/ml. Is that true and if it is, is it splenic or BM DCs?

Hi Ethan. I do often polarization assays with pure CD4+CD25- T-cells from splenocytes+lymph nodes of OT-II mice, and bone marrow-derived dendritic cells from C57BL/6 mice. For my set-up I have to use 1mg/ml of OVA (yes, it's quite a lot) . In contrast, if I use OVA peptide only 100 ng/ml is enough.

I have never tried to activate CD8+ cells, but I would guess that it would take something also in the range of mg/ml. If you don't find any protocol that suits you, you would need to titrate your OVA. Important tip, look for endotoxin-free OVA! (no traces of LPS)

I would like to ask if I would like to use OVA whole protein to pulse DC, how long normally you need to incubate with DC please? I used 18 hours, 05 ug/ml OVA. In addition, I would like to know the differences between pulse with OVA RNA and OVA protein. Could anyone give some suggestions to me please? Thank you.

Guangzhi Zhang , maybe it is too late for this response, but overnight pulsation with full OVA is totally enough as you did. Some people use very high concentrations of OVA = 500 ug/mL. You can downscale it and I 50-100 ug/mL would work, although this is quite high in comparison with pulsing with SIINFEKL peptide only. But if you go lower like 10 ug/mL, the T-cells won't proliferate really efficiently. The co-incubation time definitely plays a role, just keep it in mind.

Sidar Aydin Sorry I did not check the researchgate in the past months. Thank you so much for your kind reply. I will consider your valuable advice. Thanks. Kind regards, Guangzhi.