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Cell scraping ruining my life - (Dec/15/2010 )

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!

-azrael201-

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

-bob1-

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

-Rsm-

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?

-azrael201-

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

-scolix-

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.

-Zymech-

azrael201 on Wed Dec 15 18:22:52 2010 said:


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.

-biotechgirl-