I am going to compare dendritic cells from WT/knock mice for their ability to activate T cells, What i am planing is to activate OT II T cells together with DCs and OVA. I am new to this field, and have no idea hoe to do that. Does any one has a protocol or reference paper? Thanks a lot for that.
Hello
find the attachment & links here . also, look at the discussion in RG may help you
Good Luck
https://www.researchgate.net/post/Does_anyone_know_a_good_protocol_to_generate_OVA_peptide-pulsed_dendritic_cells_DC_in_vitro
http://www.jimmunol.org/content/185/6/3426.full
http://www.nature.com/mi/journal/v5/n2/full/mi201161a.html
http://www.pnas.org/content/103/31/11695.full
This method for spleen APC or splenic DC driven proliferation of transgenic CD4 T cells is described in the following paper:
Gabrysova, L., Nicolson, K.S., Streeter, H.B., Verhagen, J., Sabatos-Peyton, C.A., Morgan, D.J., and Wraith, D.C. Negative feedback control of the autoimmune response through antigen-induced differentiation of IL-10-secreting Th1 cells. Journal of Experimental Medicine (2009) 206: 1755-1767
Purified CD4+ T cells were isolated from spleens by magnetic separation using mouse CD4 (L3T4) MicroBeads (positive selection), CD4+ T cell Isolation Kit (negative selection) or CD4+CD62L+ T cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Splenic DCs were isolated from syngeneic mouse spleens using collagenase IV (Worthington) and DNAse I (Roche) followed by magnetic separation with CD11c MicroBeads (Miltenyi Biotec).
CD4+ T cells were cultured at 5x104 per well in complete RPMI medium (RPMI-1640 medium supplemented with 20 mM Hepes Buffer, 50mM 2‑Mercaptoethanol, 100 U/ml penicillin and 100 ug/ml streptomycin sulphate, 4 mM L-Glutamine and 5% heat inactivated fetal calf serum) in round-bottomed 96-well plates at 37°C and 5% CO2 humidified atmosphere in the presence of 1x105 per well autologous, irradiated CD4‑depleted splenocytes as APCs. Alternatively, splenic DCs were used as APCs ranging from 80:1 to 10:1 ratio of CD4+ T cells to one DC. Peptide antigen ranging from 0.1 to 100 mg/ml was added to cultures where indicated. After 72 h, wells were pulsed with 0.5 mCi [3H] thymidine overnight and the incorporated radioactivity was measured on a liquid scintillation beta‑counter (1450 Microbeta; Wallac).
It is very simple, you just co-culture DC:T cells at a 1:10 ratio (10^5 T cells per 96-well) and can look at thymidine incorporation at d5, you can alternatively prelabel the T cells with CFSE or use BrDU.
Set up culture plus and minus OTI peptide, so you can see antigen-specific proliferation. Looks like there is more detail in the response above.
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