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 lysis


  • Please log in to reply
4 replies to this topic

#1 beny

beny

    member

  • Active Members
  • Pip
  • 19 posts
2
Neutral

Posted 10 June 2011 - 02:28 AM

hi friends
i am facing this problem for quiet long.
when even i make slides for confocal and go for analysis my cell lyse.
first i do all my experiment on tubes as they are suspension culture.
3 % paraformaldehyde fixation and keep it in 4oC till i collect all my samples.
then renationalisation with methanol at -20 for 10 min or with triton x 100 0.5 % 10 min 4 oC
then blocking followed by antibody treatment.
then i wash with PBS and add 8ul of mounting media on slide and over it keep 20ul of cell suspension.
i check in microscope they are fine .will keep the coverslip on top of it and seal it.check them again cell are ok .but if the same thing i check after 1 hour cell get lysed.
what can be the reason .
please help its getting into my nerves.
Beny

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,830 posts
414
Excellent

Posted 12 June 2011 - 03:56 PM

How long are you keeping the cells. Fixing and storing at 4 deg C in PBS will only work for a week or so.

#3 beny

beny

    member

  • Active Members
  • Pip
  • 19 posts
2
Neutral

Posted 12 June 2011 - 08:56 PM

i am keeping for only 2-3 days...
i think the cell are getting crushed when i put the coverslip.
how can i avoid that

#4 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,830 posts
414
Excellent

Posted 13 June 2011 - 05:01 PM

They shouldn't be crushed by the coverslip, unless you happen to slip and cause the coverslip to move sideways while pressing down.

#5 beny

beny

    member

  • Active Members
  • Pip
  • 19 posts
2
Neutral

Posted 14 June 2011 - 03:15 AM

as the cells are suspension cells when i keep the coverslip it slides a little bit while sealing it .
how can i avoid that is there any other way to keep the coverslip so that it doesn't slide




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

©1999-2013 Protocol Online, All rights reserved.