Hello all, First, a general comment . . . saying that annexin V labeling without a viability indicator is useless (even misleading) is a bit too strong. Cells that are "dead" at the start of the apoptosis induction (assuming there is such an event) may not have become so by apoptosis, and it's important to be aware of this . . . i.e., this contributes to noise in your assay. However, this may not be important for all applications, where one, for example, might be concerned with total apoptosis regardless of time. Adding a viability marker here does make the measurement cleaner, and perhaps easier to interpret. Regardless, it is important to know that detecting apoptosis by annexin V must be validated by a companion method, and everyone should first detect apoptosis by morphology as their first step. Peter, Regarding your questions: 1) While Ficoll separation may eliminate much of the "dead" population prior to labeling, I would still expect some Annexin+/PI+ cells. 2) Yes, as apoptotic cells shift in their light scatter properties. This shift is a time/progression dependant process, so position of apoptotic cells in the FS/SS distribution may be difficult to predict. Correlating your apoptosis measurement parameter with light scatter parameters can help with this. MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Flow Core 7416 CCGC 0946 (734) 647-3216, fax (734) 936-7376 kukuru@umich.edu >>> <PLopez@adarc.org> 03/21/01 05:02PM >>> Hello, I have two questions related to the Annexin V apoptosis thread: 1- In human PBMC preps, would the Annexin V (+), PI (+) fraction go down the drain if the prep was made using a Ficoll separation? 2- Is the Annexin V (+), PI(+) fraction something that might be unintentionally gated out if the light scatter gate is too tight ? Peter Lopez The Aaron Diamond AIDS Research Center 212.448.5188 (office) 212.448.5159 (fax) 212.448.5190 or 5110 (lab)
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