Lipofectamine transfection, Flow sorting & cell viability??

Sze Wah Agnes CHAN chansw3 at hkucc.hku.hk
Wed Mar 19 22:46:11 EST 2003


Dear all, greetings!

I've done Lipofectamine transfection on an established tumor cell line.  
24-hrs post-transfection I've flow-sorted the GFP+ cells and plated them out.  
My questions are:

1. After transfection, most of the usually attached and spread out cells 
rounded up.  Many became floating but most, though round-up, remained 
attached.
Is this normal?

2. I've aspirated the floating cells (thinking they're likely dead) and 
flow-sorted only the attached cells.  Normally these cells will all attached 
and spread out overnight.  However, the transfected and flow-sorted ones 
didn't with only countable few cells attached overnight.  I did notice some 
apoptotic bodies and granular "dying-looking" cells among the floating 
population.  However the proportion of them was small.  Then I did a quick 
trypan blue staining of the floating cells and saw close to none blue dead 
cells.  ??!!  Is it usual that it may take longer for the cells to attached 
after the above processes?  Likely due to damages of the cell membrane?  If 
so, any idea how long it takes for the cells to recover?

Many thanks for advice.

Agnes Chan
Post-doc Fellow
Department of Pathology
The University of Hong KOng

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