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How to fix suspencion cells for DAPI detecting under the microscope? - (Apr/27/2009 )

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!

-Sumpf-

if you have access to a cytospin, this is a centrifuge that will allow you to spin suspension cells onto a slide.

-aimikins-

No, I don't have access :-(

-Sumpf-

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.

-little mouse-

little mouse on Apr 28 2009, 10:40 AM said:

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

-Sumpf-

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.

-little mouse-

little mouse on Apr 28 2009, 02:48 PM said:

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

-Sumpf-