Hi Jeff, There are a number of tips to prevent cell clumping. Firstly, if they are adherent or even if they grow in suspension but in clumps, make sure that they are in as good a single cell suspension that you can make before fixation. Inadequate ethanol fixation is a primary cause of clumpage - its a good idea to vortex while fixing (careful though if looking for apoptotic cells as they could explode at this point!). Also make sure that the pellet is as free as possible of residual PBS before adding the ethanol. Secondly you could try using either EDTA or a low level concetration of DNase in your PI solution as it is DNA from blown-up cells that can cause clumps as well. Also it can be a good idea to filter the cells through a 60-70um mesh to get rid of the larger lumps (although clearly this will lose cells). Finally, if they are someone elses samples, make sure you watch them as they prepare them! Good luck, Derek On Mon, 23 Oct 2000, Jeff Barry wrote: > A colleague is trying to look for apoptosis using the sub G1, Propidium > Iodide, method however, his cells stubbornly refuses to form single cells > and continue to aggregate causing our FacsCalibur to block every time we put > the samples on. Can anyone relieve our misery by suggesting any sample > preparation tips that will prevent cell clumping. ************************************************************************ Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3100 Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk London, UK mobile: 07790 604112 Web Page: http://www.icnet.uk/axp/facs/davies/index.html In tenebris lux *************************************************************************
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