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Eliminate erythrocytes from culture


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#1 Rute

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Posted 17 August 2012 - 02:03 AM

Dear all,

I am culturing primary pancreatic tissue and after digesting the pancreas there is always a portion of erythrocytes in the culture, no matter the number of time I wash the tissue before. I know that they will evenually be eliminated but, is there a way of acelerating the process?

I have heard that NaCl eliminates erythocytes. As anyone ever tried eliminating them from primary cultures? If so, I would very much appreciate any advice you might give me.

Kind regards,
Rute

#2 Rute

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Posted 17 August 2012 - 02:07 AM

Correction, not NaCl... NH4Cl.

#3 leelee

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Posted 17 August 2012 - 07:04 AM

It is routine for people to use red blood cell lysis buffer when preparing primary cells. I don't have our recipe on my lap top, but if you google "red blood cell lysis buffer" you should find plenty of recipes.

There is probably some on this protocol online website too :)

#4 rhombus

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Posted 17 August 2012 - 07:55 AM

Dear Rute,

The Red blood cell (RBC) lysis buffer used by researchers isolating Neutraphils, platelets, Monocytes from whole blood is:-

156mM NH4CL
10mM NaHCO3
0.1mM EDTA

The cells are incubated in the above lysis buffer ON ICE for 15 minutes in order to lyse the RBC's.

However if you are trying to reduce RBC contamination from your pancreatic tissue, this method MAYBE suitable IF you are not for example wanting to grow cells in culture. 15 minutes on ice will shock most primary cells and will probably kill most of them....the ones that survive will not represent the majority....i.e you will clonally select the cells able to survive low temperatures.

If you are doing cell culture the normal procedure is to isolate the primaries and them wash off the unattched RBC's, leaving the attached primaries on the TC plastic.

We really need more information about what you intend to do with the Primary tissue?

Hope this is useful

Kindest regards

Uncle Rhombus

#5 Rute

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Posted 20 August 2012 - 05:27 AM

Thank you all for your help.

I pretend to culture primary pancreatic cells, The problem is that I am starting a suspension culture and doing a first step of attached conditions (which would allow me to wash off the unattached RBC's) may compromise my culture.

I will try the treatment on ice, and take into account that I am selecting for a more resistante population. However, if you think that there is an alternative, please do tell me.

Thank you very much.

BW,
Rute




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