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help with cybrids needed


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#1 rho0

rho0

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Posted 28 December 2010 - 10:56 AM

Hi!
I have a question concerning the cybrid selection.
I performed a fusion experiment of rho0 cells (with dysfunctional mitochondria) of neuroblastoma SH-SY5Y cells with platelets.
I checked before that rho0 cells have no mitochondrial DNA but now I try to distinguish the cells thet fused with platelets from those that did not (in the same T75 plate). According to the protocol that I got, I kept the cells after fusion in the same complete medium (DMEM with high glucose, uridine, sodium pyruvate, antibiotics) for 3 weeks. Then I tried changing the medium - I replaced the rho0 complete medium with the same DMEM (high glucose) with 10%FBS and antibiotics but without uridine and pyruvate. So that those cells without functional mitochondria should die.
And here are my problems: in the negative control plate (mock fusion), the rho0 cells do not die in the selection medium. :blink: So I cannot tell if the plate containing fused platelets with rho0 is going through selection when rho0 are happily growing in the selection medium (since two weeks).

So, does anybody have experience with cybrids - how did you select them after fusion from the unfused cells? Why changing medium conditions doesn't work?

Thanks in advance for your help!




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