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 Fixation


  • Please log in to reply
5 replies to this topic

#1 ACN

ACN

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 30 posts
2
Neutral

Posted 21 July 2011 - 06:21 AM

Hello I am new at this so forgive me if this is a lame question.

I am trying quantify a virus (MCMV, Smith strain). To do this I am performing plaque assays for the pfu.

Protocol:

mCMV virus Titration

Set up NIH 3T3 cells in 6 well plates (500k cells/well)
Next day, whe cells should be 90% confluent
Thaw and sonicate virus for 6 seconds twice. Store on ice
Make a series of 10-fold virus dilutions --- 10-1 to 10-7
Aspirate media from each well and add 2ml of diluted virus to each well.
Add 2mls of media (no virus) to one well for negative control.
Spin the plate at 100g for 20 min, rotate the plate and spin again at 100g for 20 min
Aspirate the innoculum and add 3ml of overlay per well

Overlay:

100ml
45ml 2X DMEM (Final 1X)
45mls of 1% LMP agarose (0.75%)
10ml FCS (10%)
4.5ml of 7.5% Sodium Bicarbonate (0.33%)

Plate a duplicate set of plates, but use an overlay with 2% FBS


Problem:
The cells keep coming off the surface of the wells, leaving me with monolayers that that are not complete. I believe the cells are coming off when I remove the agar (agar by placing the plate in hand and then with a wimping motion the agar is flung into a beaker of bleach). I feel as the agar is removing parts of the cell surface as it is removed. This does not allow for a correct count of the plaques.

Question:
Can I do something that will fixate the cells? I have heard of using formaldehyde as a crosslinking fixative (acts by creating covalent chemical bonds between proteins in tissue. This anchors soluble proteins to the cytoskeleton, and lends additional rigidity to the tissue. -Wiki)
So if anyone has experience with this please get back to me.
Thanks!

#2 bob1

bob1

    Thelymitra pulchella

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

Posted 21 July 2011 - 03:49 PM

If you are just wanting to visualise the plaques, you can see them by illuminating the plate from the side and holding the plate over a dark background. Otherwise, you can add neutral red to the overlay, you will get clear spots developing where the dye is excluded by the dying cells.
  • ACN likes this

#3 leelee

leelee

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 652 posts
53
Excellent

Posted 21 July 2011 - 09:52 PM

I also work with MCMV.
We fix and stain our plaque assays using methylene blue with 10% formaldehyde. I've posted your protocol below, and put in where mine differs:

Set up NIH 3T3 cells in 6 well plates (500k cells/well) [i]I do mine in 24 well trays, less cells to use, more dilutions per plate etc[/i]
Next day, whe cells should be 90% confluent I wait for my cells to be 100% confluent
Thaw and sonicate virus for 6 seconds twice. Store on ice don't know why you are sonicating here?
Make a series of 10-fold virus dilutions --- 10-1 to 10-7
Aspirate media from each well and add 2ml of diluted virus to each well. I use 200ul for a 24 well
Add 2mls of media (no virus) to one well for negative control.
Spin the plate at 100g for 20 min, rotate the plate and spin again at 100g for 20 min I don't do this, are you trying to centrifugally enhance here? Why are you centrifuging? I incubate for 1 hour at 37C
Aspirate the innoculum and add 3ml of overlay per well

Overlay: I use methylcellulose rather than agarose

100ml
45ml 2X DMEM (Final 1X)
45mls of 1% LMP agarose (0.75%)
10ml FCS (10%) This is too high, you don't want your cells to be growing once inoculated with virus, you should have a final concentration of 2% instead
4.5ml of 7.5% Sodium Bicarbonate (0.33%)

Plate a duplicate set of plates, but use an overlay with 2% FBS why?

after incubation for 3-5 days, we add about 1ml of the methylene blue with 10% formaldehyde, leave at RT overnight then wash off (with the overlay) the next day

Edited by leelee, 21 July 2011 - 09:53 PM.

  • ACN likes this

#4 ACN

ACN

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 30 posts
2
Neutral

Posted 02 August 2011 - 02:46 PM

Thank you both for the help I will try all the tips.

#5 ACN

ACN

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 30 posts
2
Neutral

Posted 04 August 2011 - 01:38 PM

I also work with MCMV.
We fix and stain our plaque assays using methylene blue with 10% formaldehyde. I've posted your protocol below, and put in where mine differs:

Set up NIH 3T3 cells in 6 well plates (500k cells/well) [i]I do mine in 24 well trays, less cells to use, more dilutions per plate etc[/i]
Next day, whe cells should be 90% confluent I wait for my cells to be 100% confluent
Thaw and sonicate virus for 6 seconds twice. Store on ice don't know why you are sonicating here?
Make a series of 10-fold virus dilutions --- 10-1 to 10-7
Aspirate media from each well and add 2ml of diluted virus to each well. I use 200ul for a 24 well
Add 2mls of media (no virus) to one well for negative control.
Spin the plate at 100g for 20 min, rotate the plate and spin again at 100g for 20 min I don't do this, are you trying to centrifugally enhance here? Why are you centrifuging? I incubate for 1 hour at 37C
Aspirate the innoculum and add 3ml of overlay per well

Overlay: I use methylcellulose rather than agarose

100ml
45ml 2X DMEM (Final 1X)
45mls of 1% LMP agarose (0.75%)
10ml FCS (10%) This is too high, you don't want your cells to be growing once inoculated with virus, you should have a final concentration of 2% instead
4.5ml of 7.5% Sodium Bicarbonate (0.33%)

Plate a duplicate set of plates, but use an overlay with 2% FBS why?

after incubation for 3-5 days, we add about 1ml of the methylene blue with 10% formaldehyde, leave at RT overnight then wash off (with the overlay) the next day


Thanks for the advice.
we sonicate to make sure any viral particles are not clumped together.
the centrifuge is used to aid the virus with attachment. we then let the plate sit in the incubator for 30-60min after centrifuging.

we have lowered the % of FCS
used methlycellulose overlay and Becto agar overlay (the Becto agar overlay seems to have killed the cells).
now we wait to see what happens

#6 leelee

leelee

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 652 posts
53
Excellent

Posted 04 August 2011 - 11:41 PM

Ok, so the sonication seems reasonable. I tend to just vortex my vial and plaque assay straight from there.

Let me know how you go :)




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

©1999-2013 Protocol Online, All rights reserved.