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BrdU incorporation question

BrdU assay

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4 replies to this topic

#1 Tabaluga

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Posted 19 February 2013 - 11:51 PM

Hi, I've got a general question on BrdU incorporation. Normally BrdU pulses in vitro are ca. 30 mins. If I incubate cells for a longer time with BrdU (e.g. 2 days), the percentage of BrdU positive cells obviously increases as more cells have divided and incorporated it during their S-phase. My question is whether it's possible that the percentage of BrdU positive cells could also decrease again with longer incubation, due to it being diluted out or something similar ?

Thanks.

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#2 bob1

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Posted 20 February 2013 - 12:05 AM

BrdU could reach a point where you can no longer detect it by dilution as the cells divide, 50% fewer brdU nucleotides will go to each daughter cell at each generation.

#3 Tabaluga

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Posted 20 February 2013 - 03:29 AM

Thanks for the reply. Maybe I'm getting it wrong, but if I assume that BrdU is present in abundance in the cell medium (I don't know if it is that stable for several days though), wouldn't 100 % have it it because it is newly incorporated at each cell division / S-phase ?
In other words, dilution might become a problem when I cultivate the cells for some time after BrdU incubation in the absence of BrdU ?

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#4 bob1

bob1

    Thelymitra pulchella

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Posted 20 February 2013 - 11:43 AM

You are correct, continued incubation should lead to incorporation at each generation. I don't know how toxic or stable it is though - there could well be some sort of degradation process going on.

#5 Tabaluga

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Posted 21 February 2013 - 11:03 AM

Ok, that clears up the matter !

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 





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