T-Cell Development by Rémy Bosselut & Melanie S. Vacchio

Author:Rémy Bosselut & Melanie S. Vacchio
Language: eng
Format: epub
Publisher: Springer New York, New York, NY

Use transgenic, knockout, and conditional expression systems.

Several considerations need to be kept in mind when analyzing negative selection. It is difficult to analyze signaling in dead cells. Dead cells disappear rather quickly, even in FTOC [9, 10]. Thus, while the loss of thymocytes provides information that is essential to understand the conditions that lead to negative selection, we need to catch them before they die to be able to say anything about where they go and how they got there. Along these lines, it is important to distinguish death by neglect and death by negative selection. Thus, we always include both positive selection and non-selection controls in our analysis of negative selection (We have had numerous discussions in our lab which one should be called the positive and the negative controls and finally settled on the term selection controls as a result).

We use several standard techniques to assess signals and analyze selection in our system that are further described in Chapter 15.For early time points (usually <48 h) immunoblot techniques can be easily used to determine the expression levels and activation states of proteins. For these assays we stimulate, pool and disrupt thymi, lyse, subject to SDS-PAGE, transfer and immunoblot (shown elsewhere in this manual).

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