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!

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!

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

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/

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

Hi!

I have spent the last few hours looking for an answer to this question and thought I'd post for future reference.

Different sources claim different things. This is basically a combination of what I've found...

If we add the whole blood to pure distilled water, all the cells will eventually burst open due to osmotic pressure. So we have to modify the procedure because we only want the red blood cells to lyse.

NH₄Cl: It makes the RBC swell, because penetration of NH3 induces a transmembrane exchange between Cl and OH ions.

KHCO₃: It increases the rate of swelling of RBC and can also serve as a buffer component.

EDTA: It binds to Mg⁺² and Ca⁺², which destabilizes the RBC membrane, but does not effect white blood cells as much, so it allows us to only target the RBC. Is also a salt detergent which will eventually regulate the acidity and osmolarity of the lysate,and protects our DNA from nucleases (by binding to the ions that serve as cofactors for these enzymes).

The main point was probably to make some kind of buffer, that is safe for other cells, but exploit the specific characteristics of RBCs, their limited ability to withstand osmotic stress. 

The raeson for EDTA addition may be also more the inhibition of degradatory enzymes released from ruptured cells, rather than to help lysis.

Hello,

some revival of this old topic.. Does anybody know a way to stop the lysis of the cells by this buffer? I use ACK buffer to lyse RBCs after Ficoll isolation of PBMCs. The thing is, that if we exceed the time of 5 minutes for lysis, the PBMCs suffer as well and I need to add trypan blue and run to another room to count cells via TC20 machine. So, I wonder how representative is the count that I get if the ACK buffer is still acting until the TC20 gives my my result in duplicate....?

Thank you in advance!

Your best bet would be to wash the PBMCs in a buffer before you start counting: Spin down the cells, remove ACK buffer, replace with PBS or similar solution (e.g. Ringer's?), then take an aliquot for counting.