Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Cell scraping ruining my life


  • Please log in to reply
6 replies to this topic

#1 azrael201

azrael201

    member

  • Active Members
  • Pip
  • 27 posts
0
Neutral

Posted 15 December 2010 - 10:22 AM

hi guys,

i am a lab noob and i realize cell scraping techniques are self-explanatory...you just scrape but my god i am working with a nonneoplastic cell line immortalized by hTert and these suckers are ruining my life. I basically want to run westerns and determine the protein levels in the cytosol and nucleus so i bought a nuclear extraction kit from ActiveMotif which instructs with HeLa cells.

My problem: I grow these cells to about 80-90% confluency in 150mm plate and according to the protocol i use 6mL of PBS/phosphatase inhibitor solution to scrape in. What i have noticed for the second time is i can't get all the cells into the solution and half the cells look smeared across the plate. i am not an ogre and scraping as if i am harvest the plastic off the plate i just go with the glide of the fluid. i am guessing half are still on the plate because after centrifuging i see only about 2 million cells (150mm plate!).

right now my bright idea to get better harvesting is to recycle the PBS/PI solution after centrifuging and rinsing the plate again or should i just have made more to continue rinsing OR should i just go ahead and just use PBS to get the rest of the cells on the plate?

the cells smeared across the plate like snowflakes are basically forfeit and i should not bother trying to "clean my plate"


THANKS!

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,784 posts
405
Excellent

Posted 15 December 2010 - 02:30 PM

Scraping physically damages the cells, you will probably find that trypsinising the cells will work better for you.

#3 Rsm

Rsm

    Post Dog

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 381 posts
8
Neutral

Posted 15 December 2010 - 11:44 PM

Yes, use trypsin if you can use it. You can also try 3mM EDTA in PBS, 37C for 5-15mins, works well for most cells. Scraping is then also more fun.

rsm
I got soul, but I'm not a soldier

#4 azrael201

azrael201

    member

  • Active Members
  • Pip
  • 27 posts
0
Neutral

Posted 30 December 2010 - 08:11 AM

i guess i am just concerned about the whole issue of internalization of trypsin affecting my protein yield

however, mechanical lysis i'd imagine is also bad for that yield. so no one scrapes their cells anymore?

I am using a protocol I guess I could replace the scrape step with trypsin instead?

#5 scolix

scolix

    a student

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 314 posts
5
Neutral

Posted 30 December 2010 - 10:13 AM

some celllines you would have to scrape them and not trypsinise them as they donot like it and will not grow eg. PC12

#6 Zymech

Zymech

    member

  • Active Members
  • Pip
  • 5 posts
0
Neutral

Posted 10 January 2011 - 09:02 AM

You, my friend, have made me laugh for the first time today.

Try trypsin,
Trypsins have an optimal operating pH of about 8 and optimal operating temperature of about 37C
wash with PBS.

#7 UBClabbie

UBClabbie

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 65 posts
8
Neutral

Posted 13 January 2011 - 04:14 PM

hi guys,

i am a lab noob and i realize cell scraping techniques are self-explanatory...you just scrape but my god i am working with a nonneoplastic cell line immortalized by hTert and these suckers are ruining my life. I basically want to run westerns and determine the protein levels in the cytosol and nucleus so i bought a nuclear extraction kit from ActiveMotif which instructs with HeLa cells.

My problem: I grow these cells to about 80-90% confluency in 150mm plate and according to the protocol i use 6mL of PBS/phosphatase inhibitor solution to scrape in. What i have noticed for the second time is i can't get all the cells into the solution and half the cells look smeared across the plate. i am not an ogre and scraping as if i am harvest the plastic off the plate i just go with the glide of the fluid. i am guessing half are still on the plate because after centrifuging i see only about 2 million cells (150mm plate!).

right now my bright idea to get better harvesting is to recycle the PBS/PI solution after centrifuging and rinsing the plate again or should i just have made more to continue rinsing OR should i just go ahead and just use PBS to get the rest of the cells on the plate?

the cells smeared across the plate like snowflakes are basically forfeit and i should not bother trying to "clean my plate"


THANKS!



Hmm i can't remember the brand, but i've used to wash twice with PBS then add lysis buffer. Scrape cells into microfuge tube and use a sonicator (pulse sonicating 30s, three times) and then spun down and removed the supernatant... all on ice of course :)

i agree, trypsin might be damaging? i'm not sure but the idea just seems sketchy.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.