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How does the RBC lysis buffer work? - (Nov/04/2010 )

Hi people! I was wondering if somebody could tell me how the red blood cell lysis buffer works. What function do the ingredients have?

I used this recipe:
1 l ddH20
8.26 g Ammonium Chloride (NH4Cl)
1 g Potassium Bicarbonate (KHCO3)
0.037 g EDTA
pH 7.3

The rumor has it that it has something to do with the Na+/K+ATPase activity, but this isn't as detailed as I would like to know how it works.
I would very much appreciate an explanation how this buffer work, or maybe a hint to where I can find litterature, or the like, on this.

Thanks!

-CTC-

Come on guys! 80 views and not a single reply! Is my question unclear? Could it be that nobody knows or doesn't any body care to help a fella out?Just a hint on where I could read about it would be very much appreciated as well!

-CTC-

The other option is that many of these 80 post viewers spent some time looking into this for you (like myself, who spent over an hour on the internet trying to find out) and couldn't find anything more detailed than what you already know..........

-leelee-

leelee on Fri Nov 12 07:43:39 2010 said:


The other option is that many of these 80 post viewers spent some time looking into this for you (like myself, who spent over an hour on the internet trying to find out) and couldn't find anything more detailed than what you already know..........


Well that is also possible I guess. I apologize to all of you nice people who tried to help me out, and I also thank you for your time and effort!

-CTC-

Sorry to revive such and old thread, but I too was interested in knowing the answer. On another forum, someone use Tris instead of potassium biocarbonate and it still worked, so I don't see how it could be from Na+/K+ pumps

http://www.scienceforums.net/topic/13565-rbc-lysis/

-Ahrenhase-

Dear CTC,

Please refer to the reference below:-


http://ir.library.oregonstate.edu/xmlui/bitstream/handle/1957/13985/Schreck.AnalysisSalmonidLeukocytes.pdf?sequence=3



The important bit is:-

Mammalian erythrocytes placed into distilled water

will swell until at some critical point the rising

pressure in the cell interior overwhelms the tensile

strength of the cell membrane and hemoglobin escapes

(hemolysis). Delano (1995) developed a

model to predict the critical set of physical conditions

that result in osmotic rupture of mammalian

erythrocytes. The model determined that the

fate of the erythrocyte in a hypotonic solution depends

on tonicity, the initial sphericity of the cell,

the initial fraction of its volume not occupied by

water, and the maximum fractional amount by

which the surface area can be stretched.



We have been using your lysis buffer (NH4CL) to remove red blood cells from Ficoll-Paque isolation of whole blood. The RBC's are an unwanted contaminant as we are after Neutrophils, Monocytes, Platelets etc. By using this lysis buffer these cells are not activated in any way as to ruin our experiments i.e. degranulation and chemotaxis of Neutraphils.

I hope this gives you some more understanding of what is involved in the hypotonic lysis of RBC's

Kindest regards

Uncle Rhombus

-rhombus-