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Troubles with leukocyte adhesion - what to do?

cell culture cell adherense leukocytes

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#1 voronnoi

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Posted 04 October 2013 - 01:16 AM

Hello!

 

I am working with living leukocytes, which I use for surface-enhanced Raman spectroscopy (SERS) and it is crucial that the cells are as still as possible in order for me to create a spectral image. The leukocytes are directly extracted from human donor blood and then fronzen in liquid nitrogen (I am not doing this procedure myself).I have been trying to attach them to the well bottom (glass) by using poly-D-lysine >300.000 kDa MW, but without success.

 

The protocol I use is the one provided by Sigma Aldrich (general, not cell type specific): 

 

1. Add 50 ml of sterile tissue culture grade water to
5 mg of poly-D-lysine.
 
2. Aseptically coat culture surface with 0.5-1.0 ml of
solution per 25 cm2. Rock gently to ensure even
coating of the culture surface.
 
3. After 5 minutes, remove solution by aspiration and
thoroughly rinse surface with sterile tissue culture
grade water.
 
4. Allow to dry at least two hours before introducing
cells and medium.
 
What am I doing wrong? Is theres something special to take into consideration when trying to adhere leukocytes?
 
I also read that a) one could wash with PBS instead of water and b ) that dissolving PDL powder in borate buffer should improve adherense for some cell types. Is any of this worth a try when working with leukocytes?
 
PS. do forgive me in case I ask obvious questions. I am a chemist by training so all this cell culturing stuff is rather novel to me. 
 
Sincerely,
A. W.

Edited by voronnoi, 04 October 2013 - 01:16 AM.


#2 Meysam_Kashiri

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Posted 04 October 2013 - 01:04 PM

Hi 

Are you sure to complete lysis of RBCs? sometimes remainig RBCs in media cause troubles in cell adherence.



#3 voronnoi

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Posted 04 October 2013 - 01:09 PM

Hi, thank you for you answer!

 

If you mean red blood cells, I cannot tell as I receive those leukocytes from another laboratory. I will ask the person preparing them however. How are leukocytes different in terms of adherence from any other common cell lines for instance? What properties should one take into consideration in general when dealing with leukocytes? 







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