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T-cell activation assay: PBMC vs purified CD3+ T-cells

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Posted 15 October 2013 - 06:33 PM



I am new to immunology and need some advice on T-cell activation assay to test the immunogenicity of a particular type of cells. From the literature, I learned that activation of T-cells can be assayed by their expression of cytotoxic molecules such as granzyme B and perforin. These assays are usually performed using purified CD3+ pan T-cells.


My questions are as follows:


1. Since whole PBMCs contain 45-70% of CD3+ T-cells, can I perform the assay using whole PBMCs and then use flow cytometry to enumerate the fractions of activated subsets of CD3+ T-cells within the PBMC population that express the cytotoxic molecules?


2. What is the minimum number of individual T-cell samples do I need to include in the study in order to make it acceptable by conventional standards?


I only have limited amounts of PBMCs and enriching T-cells by any method is not really an option for me. Also, commercially available T-cells are quite costly and I don't have the budget for them unless the number of individual samples that I need is small.  


Any advice is greatly appreciated. Thank you.





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