Todd, Would using propidium iodide (PI) help? We use PI to eliminate dead cells from monodispersed preparations from mouse spleens when we separate B-cells for hybridoma preparations. All dead cells will take up the PI and yield high fluoresecence in the FL3 detector if you are using 488 excitation. Thus, you can gate the dead cells out. I will give you more specifics, if you are interested. Delynn Moss Division of Parasitic Diseases CDC -----Original Message----- From: Todd Belanger [mailto:tbelanger@vaccinex.com] Sent: Wednesday, September 18, 2002 11:04 PM To: cyto-inbox Subject: Dead cells and sorting Hi fellow sorters: I am starting to sort samples that are infected with a viral vector that tends to kill the fells over time. as a result when the samples arrive for me to sort there are as many as 20-50% dead cells in the sample. The staining/prep of the cells is a lengthy process and does contribute to the lack of health of the samples. Adding a lengthy process to remove dead cells after the samples have been stained may not be feasible due to the time required to prepare the cells in the first place. I know that this is not good for the sort and would like some advice as to what to do. I haven't had to deal with such death in any of my other sorts and have had good luck with those with decent yields and purities. Now, with the dead cells included in the samples my purities are awful ( it almost looks like I didn't sort teh sample!). I am running a Vantage w/ Diva using the same settings (using Accudrop) I used to sort cells that are not infected with a virus and with similar percentages of positive events in the samples. The samples with no virus only had 5-10% dead cells at most. Samples are filtered before sorting. How does the dead cells and debris in the sample effect the sort? Any suggestions on how to easily clean up the samples? All suggestions and help are appreciated. Thanks again, Todd Todd J. Belanger Cellualr Immunology Vaccinex, Inc.
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