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HEK 293 Synchronization


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#1 Patience

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Posted 21 July 2009 - 11:18 AM

I am attempting to synchronize HEK 293 cells through serum deprivation (DMEM is the medium). When I analyze the results on a flow cytometer, however, the results do not show that a synchronization has taken place. I am using propidium iodide as dye and the FL2-A channel of the blue argon laser (488 nm) with a 620 nm filter (the emission maximum of propidium iodide is around 617 nm) for analysis. Unfortunately, the serum-deprived samples have nearly as much of a second peak on the graph (representing the cells in mitosis) as the controls. Thinking that perhaps serum deprivation was not synchronizing the cells, I used Invitrogen's CyQUANT in order to determine cell proliferation after removing the serum. Sure enough, the HEK 293 cells had synchronized. Does anyone know what the problem could be with the flow cytometer analysis? I thought the alignment of the laser might have something to do with it, but why would only the serum-deprived samples have been affected (there was nothing wrong with the controls' graphs; only the supposedly synchronized cells showed an unnatural number of cells in mitosis, especially if they're supposed to be synchronized).

Thanks,

P

#2 miBunny

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Posted 21 July 2009 - 06:04 PM

How long after serum starvation were you performing the analysis? If it was too early, you will still see cells in mitosis. Once a cell has passed a certain point in the cell cycle, they are committed to completing the cycle. Hopefully, you are just looking at your cells a bit early.


I am not familiar with the Invitrogen kit. What exactly does it measure?

#3 Patience

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Posted 22 July 2009 - 05:34 AM

I had two time points, one at 48h and one at 72h. I think it's got to do with bizarre flow cytometer alignments and gains.

The CyQUANT kit simply consists of a dye that attaches to cells' DNA and emits a certain fluorescence that can be read by a microplate fluorescence reader. Here's the link: http://www.invitroge...ion-Assays.html




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