Culturing PBMCs with overlapping peptides derived from the sequence of the tumor antigen and IL-2 plus IL-15 prior to analysis by flow cytometry or ELISPOT assay appears to be quite sensitive, but can also induce false positives by stimulating naive T cells. Direct ex vivo methods appear to not be sensitive enough and the use of labeled multimers is limited by HLA type and the ability to identify epitopes.
It depends on the T cell type and the response you expect. For cytokine release by activated T cells elispot or FACS-based cytokine secretion assays are good choices. For antigen-induced proliferation of helper T cells the radioactivity based Tritium incorporation-assay or for cytotoxic T cells killing assays (Cr-release or non-radioactive alternatives) can be performed with few cells in 96 well plates. Other options are the detection of activation markers or to look for induction of signaling pathways by immunoblotting, i.e. phosphorylation profiling.
ELISPOT is the most sensitive method for this, especially if the cell number is limited. Try to account with controls for false positive results using background controls
What about flow cytometry? You do not need many cells and it is very sensitive
It depends on the T cell type and the response you expect. For cytokine release by activated T cells elispot or FACS-based cytokine secretion assays are good choices. For antigen-induced proliferation of helper T cells the radioactivity based Tritium incorporation-assay or for cytotoxic T cells killing assays (Cr-release or non-radioactive alternatives) can be performed with few cells in 96 well plates. Other options are the detection of activation markers or to look for induction of signaling pathways by immunoblotting, i.e. phosphorylation profiling.
Sorry me again, I forgot to read the main text body of your question. To exclude activated naive T cells this is possible with the FACS-based cytokine release assay since you can additionally stain for subset specific T cell markers.
I would probably consider qPCR (very sensitive for mRNA up/down-regulation of inflammatory markers), ELISPOT (measure soluble mediators/cell; very sensitive), for ELISA I am not entirely sure but it s worth a trial (use little medium so that the S/Ns are concentrated and therefore your sample values fall in the linear part of the standard curve) and presumably proliferation with any common reagent (e.g. CFSE, BrdU, etc.). For flow cytometry I would recommend a population of at least 3x10e5 cells to be on the safe side in terms of accurate representation.
Hello, elispot is clearly very sensitive, but you cannnot stimulate >24 to prevent naive response (best ifn-gamma as thi sneeds demethylation after cell division which take time for naive cells). The most sensitive method in our hands is rt-pcr (up to 1/1,000,000) after 24 hr stimulation by peptide pools. Best regards
dieter
I would consider direct ex vivo intra-cellular cytokine staining for MIP-1beta and CD107a (for CD8+ T-cells that is). These are two very sensitive read-outs we use a lot in the lab. And as Jochen pointed out you can exclude naive cells with adequate phenotypic markers.
Agreed with what has been said above, especially with flow cytometry-based assays. CD107a is quite sensitive and you need to incubate the cells only for a few hours (2-4 h). You may use around 5e4 cells or even less (2-3e4). Some even add directly the antibody to the co-culture. CD69 upregulation might also work if you do a 10-16 hrs co-culture. Good Luck
I would provide the same answer. Anti-CD3 antibody exposure may be used before sorting. qPCR using T-cell subset-specific primers may be another method.
real-time RT-PCR for cytokines of interest, i.e. IFNg, and ELISPOT are indeed enaoygh sensitive. BrdU also is fast (1-2 hours) and shows minute amounts of proliferating cells in FACS.
Thanks everyone for your responses. Real time RTPCR may be the best solution in this case. Can anyone point me to a publication that could help me with the method?
We have several types of work using real-time RT-PCR in T-cells. You may want to check the methodology in one of our publication in J Immunol 2009, 182: 102-110 (see methods to fig 6)
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