1. The reason for using isotype controls in a flow cytometry experiment (or any other experiment for that matter) is to control for any non-specific binding of the immunoglobulin to the cells. There can be significant binding of immunoglobulin to macrophages, monocytes or granulocytes for example because of the expression of receptors for the Fc portion of the molecule. Even if you don't have that kind of binding there is some degree of sticking on any cell type. If you compare cells that are completely unstained to those that are exposed to isotype control you'll see that there's a difference.
I have just entered into immunology research and I need to know the following: Please try to help me by answering the following questions if you have expertise.
1. What's the reason behind using isotype controls in an experiment? I need the mechanism behind the usage of isotype controls in experiments (often when we go for flow cytometry)?
2. What is the principle of using IL-4 and GM-CSF to generate DCs from monocytes (i.e. for generating MDDCs)? I understand IL-4 is an anti-inflammatory Th2 cytokine!
3. What does PolyI:C do to make DC mature?
2. Good question. It works but I'm not sure anyone really knows why or how. Some say the IL4 suppresses CD14 expression but others say that GM-CSF is enough. I think of monocytes as a non-activated myeloid cell that can differentiate to macrophage or DC depending on the stimulus.
3. PolyI:C activates DC through a TLR. Can't remember which one - TLR9 I think. This mechanism probably arose as a way of responding to viruses via the presence of nucleic acid.
Hope all that is helpful.