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How to fix suspencion cells for DAPI detecting under the microscope?


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#1 Sumpf

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Posted 27 April 2009 - 03:17 AM

Hello,

I should transfect THP-1 cells with GFP and use different transfection agents. For this I should quantify the result with DAPI. So, how can I fix THP-1 cells, which are in suspenion, for this?

Thank you for your attention!

#2 aimikins

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Posted 27 April 2009 - 08:04 AM

if you have access to a cytospin, this is a centrifuge that will allow you to spin suspension cells onto a slide.
"it is a miracle that curiosity survives formal education" -A.E.

#3 Sumpf

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Posted 27 April 2009 - 10:10 PM

No, I don't have access :-(

#4 little mouse

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Posted 28 April 2009 - 12:40 AM

did you try polylysine coated slides? let the cells sediment on the glass (put a drop of 200 ÁL of cells and let them sediment around 10 min if I remember well), they will attach. then you can wash, label, and fix.

Edited by little mouse, 28 April 2009 - 12:41 AM.


#5 Sumpf

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Posted 28 April 2009 - 02:40 AM

did you try polylysine coated slides? let the cells sediment on the glass (put a drop of 200 ÁL of cells and let them sediment around 10 min if I remember well), they will attach. then you can wash, label, and fix.


I'll try thx

#6 little mouse

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Posted 28 April 2009 - 04:48 AM

But I have a suggestion:
if you want to see GFP in THP-1 cells, it would be easier to check this by flow cytometry.
transfect your cells, then fix them with 0.5 % formaldehyde and analyse on a flow cytometer. You will see very fast the percentage of the transfected cells, and the intensity of the fluorescent per each cell.

#7 Sumpf

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Posted 28 April 2009 - 05:41 AM

But I have a suggestion:
if you want to see GFP in THP-1 cells, it would be easier to check this by flow cytometry.
transfect your cells, then fix them with 0.5 % formaldehyde and analyse on a flow cytometer. You will see very fast the percentage of the transfected cells, and the intensity of the fluorescent per each cell.


Even better than slides. Thx




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