When you perform LRC-TriCEPS for target identification are cells alive?
Yes, the cells are alive under near physiological conditions.
Do you need to genetically manipulate the cells?
No genetic manipulation (eg. ectopic expression) is needed.
Can you work with stimulated cells (i.e. activated T-Cells)?
Yes, in fact we encourage to find the best growth condition for the cells of your interest.
I have developed a complex way of growing cells to respond to my ligand of interest. Can I perform an LRC-TriCEPS experiment?
When cell culturing is not easily transferable, Dualsystems provides the TriCEPS material and the customer performs ligand coupling, receptor capture and collection of cells. The frozen cell pellets are then shipped to Dualsystems for further processing.
I do not know which cell type / cell line expresses the unknown target(s) of my ligand. Can you help?
Yes, with the Flow-TriCEPS kit you can couple your ligand of interest to a TriCEPS Version that contains a biotin. The biotin can then be used in flow cytometry to visualize the binding of your ligand to the cells. You can use the system to screen different cell types/ cell lines for the expression of the unknown target(s).
How much material do I need from my ligand of interest to perform one LRC-TriCEPS experiment?
In average, you need 300microg for a standard experiment using protein/antibodies as ligand. In case you have limited amounts of ligand available, we could design a customized experiment for you and then 100microg might also be enough. For peptides and small molecules, drop us a line.
How many cells are needed for one LRC-TriCEPS experiment?
The exact number of cells needed for one LRC experiment depends on the cell line. As a rule of thumb, a cell pellet with a volume of 50-100 microl is needed per sample. For most adherent cell lines that corresponds to 10-20×10^6 cells per sample. In a typical LRC-TriCEPS experiment, at least two treatment arms are performed in parallel: one with the ligand of interest and a second with a control ligand (that is, a ligand with a known target). Both arms are performed in triplicates to allow statistical analysis therefore roughly 60-120×10^6 cells in total are required.
In the case of primary cells from humans or animals (i.e. primary T cells) where the number of cells may be a limiting factor, we recommend performing the treatment arms in singletons but analyzing at least 3 different donors.
Can I use primary cells (i.e.T-cells) for my LRC-TriCEPS experiment?
Yes, from one donor it is expected to receive enough cells to perform an experiment with the ligand of interest and one control. As the number of cells per T-cell donor is not very large, the experiment is performed in singletons and triplicates are performed on the level of LC-MS/MS measurements. We recommend to perform an experiment on at least 3 different donors to see donor to donor variation.
I have adherent cells, is it possible to perform an LRC-TriCEPS experiment?
Yes, you can either detach the cells by adding EDTA or scraping prior to treatment with TriCEPS coupled ligand. However, we advise to add the TriCEPS coupled ligands directly to the plate so that the expression pattern of the cells is not altered during detachment of cells.
On what type of cells can you perform an LRC-TriCEPS experiment to identify the targets?
Any cell type that has glycosylation machinery.