We are using the following conc.
1L ddH20 8.26 g Ammonium Chloride (NH4Cl) 1 g Potassium Bicarbonate (KHCO3) 0.037 g EDTA pH 7.3
Why the altered tonicity, osmolarity and buffer affect only RBCs? Or does it has something to do with MSCs?
Because it is RBCs lysis buffer
RBC lysis buffer Thats Why it lonly affects the RBCs.
Cheers !
Thanking you
@ Sunil Kumar, that I know. My specific question was why the action of NH4Cl on plasma membrane, buffering action of KHCO3 and chelating action of EDTA work only on RBCs and not on MSCs? If you have the answer please shed some light on it.
I find this on the web
NH4Cl is the active agent in most RBC lysis buffers. The solutions are typically iso-osmotic (so as to not destroy all the other cells), so there is something about this situation that throws the osmotic balance inside the RBCs out of whack. NH4 is in equilibrium with NH3. The NH3 diffuses across the cell membrane. Inside the cell, the ammoniumammonia equilibrium is re-established: (1) H2O + NH3 NH4 + OH If you get rid of the OH, the reaction will be driven to the right. RBCs (and not leukocytes) have a membrane Cl/HCO3 exhanger. With the Cl in the lysis buffer coming in, the HCO3 is going to exit the cell. Inside the cell, there is the carbonic anhydrase catalyzed equilibrium: (2) CO2 + OH HCO3 This equilibrium will be shifted to the right inside the cell as the HCO3 is exchanged for Cl. This means OH is being consumed, which drives equilibrium (1) towards the right. The overall result is net influx of NH4Cl into the RBCs which results in increased osmotic pressure until the cells burst from the water influx. Afterward, the PBS would be used to wash away the NH4Cl. I think that the edta is just to inactivate proteases and nucleases released on cell lysis. I think that the cell walls of most other cells than rbc are just stronger so will burst eventually . I used to lyse rbc sticking to white cells prior to ebv transformation by washing the cells in water until the colour changed then adding 2X saline and the white cells always seemed to survive and transform well so they are probably quite robust
I hope this lnke is usdfull for you
Article Measurement of the nonlinear elasticity of red blood cell membranes
Article Erythrocyte lysis in isotonic solution of ammonium chloride:...
The lysis buffer may harm peritoneal macrophages (at least in my experience). So I guess it is not safe for any type of immune cell.
Veljko Blagojević , what exactly do you mean by saying that the RBC lysis is harming the peritoneal macrophages? Do they burst as well and disappear in the supernatant, or do the surfacemarkers change, or function, or...? (Mice or human?) Thanks!
Roselien Vandecasteele I didn't observe them under the microscope, but they cease production of NO or cytokines in culture (it goes down about 10x), and when inspected on flow cytometry, the macrophage gate is severely depleted (and the FSClow/PI+ dead cells gate greatly enriched), while the lymphocyte and granulocyte gates are mostly normal. I only worked with rat cells, I don't have experience with mice/human peritoneal cells.
Veljko Blagojević Thank you for the quick response! I will check this in my experiments as well! Thanks!
Roselien Vandecasteele No problem. Would you be so kind to notify me of your results in this particular problem? I would appreciate it.
Hi Dayanidhi,
The concept of Cl/HCO3 that Paul Rutland explained is correct. The only thing is that Cl/HCO3 exchanger appears on the surface of mature RBC in the late stage of development. Band 3 is a protein responsible for exchanging Cl/HCO3 which comes to the surface of RBC membrane during membrane remodelling. Since leukocytes and immature RBC do not express Band 3 on their surface or express in lower level, the do not get lysed by the buffer.
This paper explains how RBC are different in different stage of development.
https://www.pnas.org/content/106/41/17413
Moa Shao We typically used 10 minutes, at 4C, with occasional stirring. I attempted even shorter periods, about 5 minutes, and the results were still not good.
Veliko, potassium efflux causes pyroptotic death in macrophages. I would try putting in at least 5mM KCl in your lysis buffer. This may save your macrophages. Also, try shorter periods at warmer temperatures.
Paul Rutland That is the best answer I've found anywhere! Thank you.
Jack Rivers-Auty Thank you for your answer, we will try it out the first chance we get. I will report back. :)
Veljko Blagojević
My lab routinely uses the formula that the OP posted (.15M AmCl, 10mM K bicarb + EDTA), and we do not have any issues with losing peritoneal macs (or bone marrow monocytes, splenocytes, or PBMCs). We do a 3-5 minute lysis at RT followed by a 5 minute spin. On whole blood, you know lysis is complete because the sup goes from opaque to clear. There is also the option of not RBC lysing your pMacs if your pull is clean enough or if you're doing a selective adhesion/bead purification to exclude neutrophils.
why different labs use different concentrations of sodium bicarbonate and disodium EDTA for RBCs lysis, despite having the same molar concentrations of ammonium chloride.
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