1. Except for the mature enucleated RBC and α-intercalated cells in the collecting duct of the kidney, no other cells in the body express the Band 3 anion exchanger and so are protected from lysis.

2. I am not sure what your application is but the simple lysis buffer is composed of mostly harmless components, so it should be fine if you keep it longer. Most, commercial buffers mention that they cause minimal effects on leukocytes. But in practice, I find that experiments in biology work well if you minimise incubations to the reccomended amount so as to avoid any degradation of protein/RNA or whatever it is that you want to assay/collect.

3. RBC lysis buffers are typically isosmotic and aim to keep all cells in solution in osmotic balance so as to avoid their rupture. The active ingredient for RBC lysis is Ammonium Chloride. The osmotic rupture of RBC alone occurs because of the prescence of an abundant membrane Cl—/HCO3— anion exhanger called Band 3 on the surface of mature RBC. Upon exposure to the lysis buffer, the chloride ions will be driven to enter the RBC via the exchanger because of the ion gradient. This will cause the HCO3— ions to be driven out by the exchanger. To compensate, the carbonic anhydrase mediated equilibrium (HCO3− + H+ CO2 + H2O) will shift to the left and increases depletion of CO2 and H20. This causes water to rush into the RBC and weaken its integrity and eventually lyse it. The ion exchange process is slightly more complicated and includes an imbalance in K+, OH− and NH3 equilibrium as well. You can check this material if you want to know more (DOI: 10.1016/j.jtbi.2007.10.016). This old nature paper explains the Ammonia/Ammonium and potassium ion balance (which I didnt talk about) very well (https://doi.org/10.1038/151252b0)

4. Yes, there are a few recipies out there. I have come across ones with Sucrosein it as well. I havent used them all. But the ACK lysis buffer (NH4Cl: 8,024 mg/l KHCO3: 1,001 mg/l, EDTA.Na2·2H2O: 3.722 mg/l )is good for lysing mouse/human red blood cells while keeping the lymphocytes intact.

1. Except for the mature enucleated RBC and α-intercalated cells in the collecting duct of the kidney, no other cells in the body express the Band 3 anion exchanger and so are protected from lysis.

2. I am not sure what your application is but the simple lysis buffer is composed of mostly harmless components, so it should be fine if you keep it longer. Most, commercial buffers mention that they cause minimal effects on leukocytes. But in practice, I find that experiments in biology work well if you minimise incubations to the reccomended amount so as to avoid any degradation of protein/RNA or whatever it is that you want to assay/collect.

3. RBC lysis buffers are typically isosmotic and aim to keep all cells in solution in osmotic balance so as to avoid their rupture. The active ingredient for RBC lysis is Ammonium Chloride. The osmotic rupture of RBC alone occurs because of the prescence of an abundant membrane Cl—/HCO3— anion exhanger called Band 3 on the surface of mature RBC. Upon exposure to the lysis buffer, the chloride ions will be driven to enter the RBC via the exchanger because of the ion gradient. This will cause the HCO3— ions to be driven out by the exchanger. To compensate, the carbonic anhydrase mediated equilibrium (HCO3− + H+ CO2 + H2O) will shift to the left and increases depletion of CO2 and H20. This causes water to rush into the RBC and weaken its integrity and eventually lyse it. The ion exchange process is slightly more complicated and includes an imbalance in K+, OH− and NH3 equilibrium as well. You can check this material if you want to know more (DOI: 10.1016/j.jtbi.2007.10.016). This old nature paper explains the Ammonia/Ammonium and potassium ion balance (which I didnt talk about) very well (https://doi.org/10.1038/151252b0)

4. Yes, there are a few recipies out there. I have come across ones with Sucrosein it as well. I havent used them all. But the ACK lysis buffer (NH4Cl: 8,024 mg/l KHCO3: 1,001 mg/l, EDTA.Na2·2H2O: 3.722 mg/l )is good for lysing mouse/human red blood cells while keeping the lymphocytes intact.

The RBCs are quite structurally fragile cells compared to PBMCs and as such they lyse quite quickly while the PBMCs can survive the transient hypotonic shock. However, adding pure distilled water to the whole blood will eventually burst open all the cells due to osmotic pressure. So as to lyse RBC more specifically, people took advantage of NH4Cl solution as a buffer that serve the purpose. RBCs are excessively permeable to NH4Cl, uptake of which results in altered pH gradient in RBCs and subsequent osmotic lysis through the uptake of chloride and water and finally in cell swelling in physiological isotonic conditions. While, PBMCs remain in ionic equilibrium with the isotonic buffer and are thus not lysed.

The other two ingredients of ACK lysis buffer are the Sodium Bicarbonate and EDTA. Sodium Bicarbonate which is basically baking soda, acts as a adjuvant in cell swelling. Baking soda in excess can be related to salt retention, including raised blood pressure and swelling in general. While EDTA addition aid in inhibition of degradatory enzymes, inactivate proteases and nucleases released from ruptured cells.

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 within the given short interval of time. Given the mechanism of action, if the cells are incubated for an extended period of time, there would be certainly bad effect on other leukocytes.

Role of pH in RBC lysis solution

I want to add another question in context of RBC lysis solution used in flow cytometry

We have been using our own lab made lysis solution containing ammonium chloride sodium bicarbonate and edta but when we keep the cells for more than 15-20mins in the solution out neutrophills convert into debris Or start to loose there integrity and shift to lower forward and side scatter.

Does pH play an important role here?

How long we can store the solution?