The first thing that springs to mind is that 7AAD intermediate staining could be revealing cells that are undergoing apoptosis. This does not seem an unlikely scenario for bone marrow cells, especially since it was fresh material. Was this sample froma healthy individual? Apoptotis in the bone marrow could be a consequence of increased haematopoiesis or a marker of disease (or the sample was handled badly!). Most cell-impermeant viability dyes, such as Propidium Iodide and Sytox Green, will reveal a dim positive population if cells are stained for long enough. This usually a consequence of cell membranes becoming more permeable as a result of ongoing apoptosis. This technique is not full proof, but can be a good indicator. Check the light scatter properties of the dim positive cells and compare that with the negative and the bright positive. Apoptotic lymphocytes do exhibit altered light scatter characteristics compared to viable and necrotic cells. Remember staining concentration is also a factor that will affect the ability to detect the 7AAD dim + cells. You could confirm this staining pattern on other lymphocyte cell lines induced to undergo apoptosis. After that, there are now many ways to measure apoptotis by flow cytometry.............. Gill Webster PhD Head of Flow Cytometry Genesis Research and Development PO Box 50 Auckland New Zealand email: g.webster@genesis.co.nz direct dial 00 61 9 3743708 fax: 00 61 9 373 2189 -----Original Message----- From: Jeff Louie [mailto:jlouie@hsc.usc.edu] Sent: Wednesday, July 18, 2001 6:30 AM To: cyto-inbox Subject: 7 AAD I have tried using 7AAD for viability using the procedure from Current Protocols in Cytometry section 9.2.3. I made the stock solution using the procedure found on 7.11.4. I am using a FacsCalibur flow routinely used for three color applications and selected the Percp setting for my analysis. The results on one bone marrow sample demostrated clear negative and positive populations, but included a moderately bright 7AAD positive population between the neg and pos population. The positive population was determined/verified by setting up a previously fixed sample, CDChex, as a positive control and the bone marrow sample used was tested on the same day it was collected. I would like to have some imput and a few alternate procedures to follow to eliminate the indeterminate populations or better understand the significance of the moderately bright 7AAD population on a fresh sample. Mel
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