Proliferation

 
An other way to measure a successful (immune) response, besides cytokine production, is to look at proliferation of the appropriate immune-competent cells. To measure this proliferation there are a few protocols:

1. incorporation of tritiated-thymidine. This thymidine analogue is build in the DNA that is newly formed. The higher the proliferative response to an immunogen, the higher the number of counts in the respective culture will be.
Disadvantage of this method is that the cell type responsible for this reaction can't be distinguished without isolation of the population at forehand. But for some reactions, like the proliferative response of lymphocytes to an antigen, other cell types are necessary. It is also impossible to discriminate how many cells originally  have reacted to an antigen. And of course, a radio-nucleotide has to be handled.

2. by labeling cells with the carboxyfluorescein diacetate, succinimidyl ester (CFDAse) cell divisions can be evaluated making use of a flow cytometer. Each daughter cell will get half the fluorescence of the mother cell. That way not only the reaction to an immunogen, but also the originally reaction population can be calculated. It is even possible to track divisions after the cells have been put back to and retrieved from an an animal. And it is possible to use the dye together with surface markers to discriminate cell populations or combine it with intracellular (cytokine) staining.

3. comparable to the tritiated-thymidine, the non-radioactive thymidine analogue bromodeoxyuridine (BrdU) can be added to the the culture. This method, making use of a BrdU specific antibody, has now been made possible to measure the DNA synthesis together with surface markers and intracellular staining.

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 Suggestions, literature and protocols are welcome at icstaining@schuitemaker.info. Copyright 2000 - 2005.
Page was last updated: 05-03-2005