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collecting cells HT1080


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#1 kamilaf

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Posted 17 June 2011 - 07:06 AM

I am trying to collect cells HT1080 (fibrosarcoma) from 24-well plate, but they are very stubborn, I washed first with PBS, and then tried to take them together with 200ul of PBS by scraping them with tip and pipetting up and down, but many cells satyed in wells. Do you know any method for taking them? The purpose is to collect them do lysis and Western Blot.

Thank you!!!

#2 GNANA

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Posted 17 June 2011 - 07:25 AM

Why dont you directly lyse them in the plate itself ???
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#3 bob1

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Posted 19 June 2011 - 04:47 PM

You could also use trypsin to lift them and then collect and lyse.

#4 kamilaf

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Posted 20 June 2011 - 07:04 AM

Yes, I thought about trypsin, you think that the concentration normally like with standard passaging, only take smaller amount to each well? Won´t it interact later with proteins?

Or lysis directly in wells, so firt washing with PBS, then incubation 30 min on ice with lysis buffer, and then collecting and centrifuging? I was afraid that to collect cells with lysis buffer because it is with detergent, so I will have foam etc.

Thanks!!

#5 bob1

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Posted 20 June 2011 - 05:07 PM

Yes, same concentration as for passaging your cells. I use about .25 ml /well of a 6 well plate, but would probably only use 0.1 ml for a well of a 24 well plate.

Don't worry about a little bit of foam, you should be able to pick up most of the cells without too much pipetting up and down.




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