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How to view the cell treated with bacteria under SEM - (Mar/16/2007 )

Im not sure im in the right place or not, but i have one question regarding this topic.

Im using INT 407 cell treated with becterial cell and want to see weather the bacteria can attach to the cells or not.

I had use inverted microscope and now i want to see under Scanning electron microscope,

In the jurnals the are using coverslip, and i want to know, do i have to use a specific cover slip?

and what is the formula to use if im using poly L lysin to coat the cover slip?

Thank you in advance tongue.gif

-zaihana-

We have done a similar thing in the past. It is easier and quicker if you have the use of a Confocal Microscope. Put the cells onto glass coverslips and infect with bacteria. Fix and stain cells, do a Z-series using the confocal. Use the 3D package to assembly all the individual images. You should easily be able to see if the bacteria are internalised or just attach to the cell membrane.

-Rhombus-

QUOTE (Rhombus @ Mar 16 2007, 04:48 PM)
We have done a similar thing in the past. It is easier and quicker if you have the use of a Confocal Microscope. Put the cells onto glass coverslips and infect with bacteria. Fix and stain cells, do a Z-series using the confocal. Use the 3D package to assembly all the individual images. You should easily be able to see if the bacteria are internalised or just attach to the cell membrane.


What would you recommend for fixing the slides? we have been using LIVE/DEAD stain so far on the CLSM but don't have to fix the material. I am looking into doing a similar experiment but don't know about fixation of cells to a slide!

Thanks

-maymunah-

QUOTE (maymunah @ Mar 28 2007, 12:57 AM)
QUOTE (Rhombus @ Mar 16 2007, 04:48 PM)
We have done a similar thing in the past. It is easier and quicker if you have the use of a Confocal Microscope. Put the cells onto glass coverslips and infect with bacteria. Fix and stain cells, do a Z-series using the confocal. Use the 3D package to assembly all the individual images. You should easily be able to see if the bacteria are internalised or just attach to the cell membrane.


What would you recommend for fixing the slides? we have been using LIVE/DEAD stain so far on the CLSM but don't have to fix the material. I am looking into doing a similar experiment but don't know about fixation of cells to a slide!

Thanks


The easiest thing to do is to grow the cells onto glass coverslips. Then put these into 6/12/24 well plates and do the bacterial infections in the wells. Then you fix and stain the cells attached to coverslips in the wells. Once this is done you invert the glass coverslips onto the glass slides ready for visualisation.
For our invasion assy we used Propridium Iodide (PI) to stain DNA, the cell nucleus but also bacterial DNA. It is very easy to use a confocal to get:
a) Bacteria that adhere to the cell membrane
cool.gif Bacteria that have invaded and can be clearly seen in the cytoplasma
c) Do a Z-series (20-30 Sections) and complile 3D to verify internalisation


N.B. It is very useful to counterstain for structural proteins i.e. FITC (green) for actin or Tubulin.

Hope this is useful.

Rhombus

-Rhombus-

QUOTE (Rhombus @ Mar 28 2007, 12:04 PM)
QUOTE (maymunah @ Mar 28 2007, 12:57 AM)
QUOTE (Rhombus @ Mar 16 2007, 04:48 PM)
We have done a similar thing in the past. It is easier and quicker if you have the use of a Confocal Microscope. Put the cells onto glass coverslips and infect with bacteria. Fix and stain cells, do a Z-series using the confocal. Use the 3D package to assembly all the individual images. You should easily be able to see if the bacteria are internalised or just attach to the cell membrane.


What would you recommend for fixing the slides? we have been using LIVE/DEAD stain so far on the CLSM but don't have to fix the material. I am looking into doing a similar experiment but don't know about fixation of cells to a slide!

Thanks


The easiest thing to do is to grow the cells onto glass coverslips. Then put these into 6/12/24 well plates and do the bacterial infections in the wells. Then you fix and stain the cells attached to coverslips in the wells. Once this is done you invert the glass coverslips onto the glass slides ready for visualisation.
For our invasion assy we used Propridium Iodide (PI) to stain DNA, the cell nucleus but also bacterial DNA. It is very easy to use a confocal to get:
a) Bacteria that adhere to the cell membrane
cool.gif Bacteria that have invaded and can be clearly seen in the cytoplasma
c) Do a Z-series (20-30 Sections) and complile 3D to verify internalisation


N.B. It is very useful to counterstain for structural proteins i.e. FITC (green) for actin or Tubulin.

Hope this is useful.

Rhombus


Thanks for this. Sorry to be so dense but is there anywhere I can a find a more detailed protocol? I am new to cell culturing and end up asking rather silly questions!

I have looked into your suggestions and found Thermonox coverslips. Are these any good? From what you have said, I should grow my cells on this type of coverslip, put coverslips into wells and then infect after there is an adherent monolayer? My questions would again be what to use for fixation after application of stains? And would it be easy enough to remove the coverslips from the wells in order to visualise?

I'm familiar with confocal things but am stuck with everything before actually using the confocal!

-maymunah-