I want to study the result of the immune response which happens when we add certain number of cells to a foreign blood, like that which happens after kidney or bone marrow transplantation.
You can performei a mixed lymphocyte reaction using donor and recipient cells together in a culture medium. Classically, you mark the donor cell with thymidine and after 7 d you count the cell proliferation by measuring radioactivity. as controls you have to use isogenic cells and third party cells.
you can't. the immune response in vivo will be the result of all sorts of signals that don't occur in vitro. so it really depends on what you want to know. if the strength of the response is all you need, then, as listed above, the mixed lymphocyte culture can give you a reasonable idea. but if you want to know more than that (like what kind of response occurs to different transplants, or the kinetics of the response, or data on the homing of cells to the target organ, or what types of cells are involved in the rejection or tolerance, then the Mixed lymphocyte reaction is not enough. So, it would be helpful to know what question you are asking.
I want to study the effect of immunological response on certain mixture of cells (like whole blood or bone marrow) to know the final conditions of attacked cells like the number and types of injured cells, which cells were most immunogenic and which were least immunogenic.
Polly is right saying about incapability to reproduce in vivo response in in vitro cultures. Really, in the MLR in vitro you can roughly estimate overall MHC mismatch between donor and recipient, which will be partially correlated with transplant rejection rate. I am surprized by your interest, because it is well known that professional APC (especially, dendritic cells ubiquitously distributed in different organs) are the most immunogenic, whereas another cell types are less immunogenic. Correspondingly, in th MLR you can easy initiate immune response to DC or monocytes, but unlikely you will see significant response to erythrocytes, epithelial cells or fibroblasts. So, you can evaluate immunogenicity measuring proliferation of lymphocytes in the MLR. Transplant rejection in vivo is more complex process in which several processes are involved 1) direct destruction of transplanted cells by CTLs and NK-cells; 2) termination of blood supply by effectors of DTH, 3) destruction of transplanted cells by activated macrophages; 4) antibody- and complement-dependent cytotoxicity, etc. These in vivo phenomena can be partially reproduced in vitro. To trace the types and fate of injured cells, you need to use any reliable molecular marker associated with transplanted cells. For example, you can use cells from GFP-transgenic animals for transplantation. Then, you will have possibility to identify transplanted cells in recipient or in in vitro culture and to determine cell types by additional staining with mAbs to lineage-specific antigens and subsequent analysis by microscopy or flow cytometry.
I agree with this discussion and Polly's comments in particular. But another option from the MLR is to use CD3/CD2 costimulation. This is another way that the principle of cognate antigen recognition with costimulatory signal leads to T cell activation. Such CD3CD28 activation is very powerful and can be achieved with commercial beads or by getting the antibodies and coating culture plate wells. In the latter, put CD3 on the plate first at 1 mcg/ml concentration for 4 hours at RT. Then you have a choice of adding anti-CD28 (cis) or putting the anti-CD28 into the culture media at the time of activation (trans). You should determine the optimal concentrations for your lab and your system. But please remember that CD3/CD28 activation is a very contrived activation and obviously not appropriate for some applications. As Polly and others note, MLR is a classic method but also highly contrived compared to a real immune response. However, a whole lot of great science has been done with both methods and it all depends on your questions and strategy.
Faez, it looks, from your one publication, that you work with humans tissues, so many of the reagents available to people who work with mice will not be available to you (unless you switch to mice : >). So let's first get clear what question you would be asking. i presume that you would be taking blood samples from a person and analyzing the cells in the samples. when you say that you want to know "effect of immunological response on certain mixture of cells", what immune response are you looking at? what cells would you add
My idea is all about bone marrow transplantation.I want to study the relationship between cell immaturity and immunogenicity. Some studies suggest that immature cells are less immunogenic and vice versa so that I need a system that's to some degree similar to the real immune system to study this. By the way, I can switch to mice if that was the solution.
The ideal model of modulator has a balanced and functional administration of cytokines and interleukins involved in cell maturation phase of innate and adaptive immune response.
The best application of stimulating the beginning of time and the end of each stage of the immune response is to understand the ideal model of tolerance.