ACK lysis buffer

ACK lysis buffer is chemically simple. Three solutes in water, adjusted to physiological pH:

Standard 1X ACK lysis buffer, per 1 litre

Filter through a 0.22 micron membrane. Store at 2-8 degrees C protected from light, or at 15-30 degrees C for products supplied at ambient storage; check the label, because commercial ACK is sold under both conditions.

Commercial formulations vary slightly, and the variation is real. Lonza's published instructions for use specify NH4Cl at 8,024 mg/L, KHCO3 at 1,001 mg/L and EDTA.Na2.2H2O at 3.722 mg/L, which is a tenfold lower EDTA content than the 0.1 mM figure that appears in most published recipes. Gibco's ACK Lysing Buffer is specified at pH 7.0-7.7 with an osmolality of 280-320 mOsmol/kg, and the ammonium chloride content is often quoted at 155 mM rather than 150 mM. None of these differences is large enough to change how the buffer behaves in practice, but if you are reproducing a published method exactly, use the recipe that method used rather than assuming all ACK is identical.

The EDTA is the component people most often ask about. It is present at a chelating, not a lysing, concentration: it binds divalent cations to prevent cation-dependent clumping and to keep the leukocyte suspension single-cell. It contributes essentially nothing to the lysis itself, which is driven by the ammonium chloride.

A note on the "10X" question. ACK is normally supplied and used at 1X. Some suppliers sell a 10X concentrate that is diluted into water before use. A 10X stock is not isotonic and must never be applied to cells undiluted.

The selectivity of ACK is not a matter of red cells being fragile. It is a consequence of two proteins that mature erythrocytes carry in enormous quantity and leukocytes do not.

Ammonium chloride in solution is in equilibrium with ammonia and protons. Uncharged ammonia crosses the erythrocyte membrane freely. Inside the cell it picks up a proton to become ammonium, consuming intracellular protons and shifting the carbonic anhydrase-catalysed equilibrium between carbon dioxide and bicarbonate. Erythrocytes contain very high carbonic anhydrase activity, so this equilibration is fast.

The bicarbonate produced is then exchanged for extracellular chloride by band 3 (AE1), the erythrocyte anion exchanger, which is present at roughly a million copies per cell and catalyses electroneutral one-for-one bicarbonate/chloride exchange. The net result of this loop, known as the Jacobs-Stewart cycle, is continuous net accumulation of ammonium chloride inside the cell. Osmotically obliged water follows, the cell swells, and the membrane fails.

Leukocytes have neither the carbonic anhydrase activity nor the band 3 density to run this cycle at any appreciable rate. They take up some ammonia and experience a transient rise in intracellular pH, but they do not accumulate solute fast enough to swell and burst within the few minutes the protocol runs. This is why timing matters so much: the difference between a red cell and a white cell in ACK is kinetic, not absolute. Leave the cells in long enough and the white cells suffer too.

Two consequences follow directly from the mechanism. First, lysis is faster at room temperature than on ice, because every step in the cycle is temperature-dependent; chilling the buffer is the most common reason lysis appears incomplete. Second, the transient intracellular alkalinisation that leukocytes experience is not biologically neutral. For functional readouts that are sensitive to cytosolic pH, such as calcium flux, oxidative burst or phagocytosis assays, density gradient separation is often a safer choice than ammonium chloride lysis.

This is the standard application ACK was designed for. Volumes below are for one adult mouse spleen.

1. Prepare a single-cell suspension. Mash the tissue through a 70 micron cell strainer into cold PBS or serum-free medium using the plunger of a syringe. Rinse the strainer.
2. Pellet the cells. Centrifuge at 300-400 x g for 5 minutes at 4 degrees C. Aspirate the supernatant completely; residual supernatant dilutes the lysis buffer and slows the reaction.
3. Resuspend in ACK. Add 1-3 mL of 1X ACK lysis buffer per spleen. Lonza's instructions give the general rule for any pellet: add a volume of ACK comparable to the volume of the pellet. Resuspend gently and completely - clumps lyse unevenly.
4. Incubate. 1-5 minutes at room temperature, mixing gently. Lonza's protocol specifies swirling the tube for 30-60 seconds. Many laboratories watch the suspension and stop as soon as it turns from opaque red to clear translucent red, which typically takes 1-2 minutes. Do not exceed 5 minutes.
5. Stop the reaction by dilution. Immediately fill the tube to at least 10 volumes with PBS or serum-free medium. Dilution, not neutralisation, is what stops ACK.
6. Wash. Centrifuge at 300 x g for 5 minutes, discard the supernatant. The pellet should now be white or pale pink.
7. Repeat if necessary. If the pellet is still visibly red, repeat steps 3-6 once. A second round is normal for spleen; a third suggests something went wrong earlier, usually incomplete resuspension or cold buffer.
8. Filter and count. Pass through a 40-70 micron strainer to remove debris and lysed-cell aggregates, then count with trypan blue and proceed.

Bone marrow follows the same protocol with a shorter incubation, typically 30-90 seconds, because the red cell burden is lower.

Buffy coat and diluted blood need more buffer relative to sample and a longer incubation, usually 5-10 minutes at room temperature with 10-20 volumes of ACK per volume of packed cells. Manufacturers differ on whole blood: Gibco lists EDTA-treated whole blood among the intended sample types, while Lonza's instructions for use state explicitly that the product is not to be used for lysing whole blood. Follow the instructions supplied with the material you actually bought.

Incomplete lysis - the pellet stays red. Causes, in order of frequency: buffer used cold or straight from the fridge (warm to room temperature first); pellet not fully resuspended, leaving clumps the buffer cannot reach; too little buffer for the red cell load; too much residual supernatant diluting the buffer; expired or repeatedly frozen buffer. Residual red cells are not merely cosmetic - they clog cytometer fluidics, distort automated counts and add debris to downstream preparations.

Over-lysis - viability drops and yield falls. The single most common protocol error is leaving cells in ACK too long. Beyond about five minutes, leukocytes begin to swell and die, granulocytes are affected first, and surface epitopes can be lost or altered enough to affect flow cytometry staining. If your post-lysis viability is below 85-90% for a fresh spleen preparation and the tissue handling was clean, the lysis step is the first place to look.

Cells clump after lysis. Free DNA from lysed cells is the usual cause. Adding DNase I to the wash step, or including EDTA in the wash buffer, will normally resolve it. Filtering through a 40 micron strainer immediately after the final wash removes the aggregates that have already formed.

Downstream enzyme reactions do not work. EDTA carried through from the lysis buffer chelates calcium and magnesium and will inhibit any divalent-cation-dependent enzyme you use next - restriction enzymes, polymerases in some buffer systems, and collagenase or dispase in particular. Wash twice in a divalent-cation-containing buffer before any enzymatic step, and never carry ACK-treated cells straight into a dispase or collagenase digestion.

Functional assays behave oddly. The transient intracellular alkalinisation described above can alter activation state, calcium handling and respiratory burst measurements. If a functional readout looks inconsistent with the flow phenotype, compare against density-gradient-separated cells from the same donor before concluding anything biological.

Contamination appears days later. ACK is a simple aqueous solution with no antimicrobial activity and no preservative. Prepared in-house from unfiltered water, or decanted repeatedly from a shared bottle, it is a convenient vehicle for contamination into an otherwise clean preparation. Filter at 0.22 micron, aliquot, and treat it with the same care as medium.

Ammonium chloride is a skin, eye and respiratory irritant and is harmful if swallowed. At the concentrations used here the solution is mild, but the solid is not, and weighing it out generates dust. Weigh in a fume hood or with local extraction, wear eye protection, and avoid inhaling the powder.

Do not mix ammonium chloride solutions with hypochlorite bleach. This is the one genuinely serious hazard associated with an otherwise benign reagent. Ammonium salts react with hypochlorite to form chloramine gas, which is toxic on inhalation. Laboratories that decontaminate waste aspirators with bleach need to be aware that ACK waste going into a bleach trap is a real chloramine risk. Dispose of ammonium chloride waste separately, or dilute heavily with water before it reaches any hypochlorite.

Contact with strong bases liberates ammonia gas for the same underlying reason.

Endotoxin matters for immunology. ACK is used almost exclusively on immune cells, which are the cells most sensitive to lipopolysaccharide contamination. A buffer prepared from laboratory deionised water of unknown endotoxin content can prime or activate the very cells you are about to phenotype, and the effect will look like biology. Use cell-culture-grade water with a stated endotoxin specification, and for functional immunology use a commercial buffer with a documented endotoxin limit.

Storage and stability. Once prepared and filtered, ACK is stable for months refrigerated and protected from light. Commercial 1X buffer typically carries a 12-month shelf life. Do not freeze and thaw repeatedly. Discard any bottle that turns cloudy or develops particulates.

ACK is not the only way to get rid of red cells, and it is not always the best one.

Hypotonic (water) lysis works on the same osmotic principle but crudely: brief exposure to distilled water followed by restoration of tonicity with concentrated saline. It is cheap and needs no reagents beyond water and 10X PBS, but the window between lysing red cells and damaging leukocytes is much narrower than with ACK, and reproducibility between operators is poor. It survives mainly as a fallback.

Density gradient separation (Ficoll-Paque, Percoll, Lymphoprep) removes erythrocytes and granulocytes together by buoyant density rather than lysing anything. It is gentler, it does not expose leukocytes to ammonium or to intracellular pH shifts, and it is the right choice for functional assays. The costs are time, cost per sample, loss of granulocytes if you wanted them, and a technique that is harder to run reproducibly at scale.

Commercial fix-and-lyse reagents used in clinical flow cytometry lyse red cells and fix leukocytes in one step. They are excellent for immunophenotyping of whole blood and useless if you need live cells afterwards.

No lysis at all is a legitimate option when the downstream method tolerates red cells - some magnetic bead isolations, for instance, do not care.

For the specific job ACK was built for, preparing viable mouse splenocytes or bone marrow cells for culture, sorting or adoptive transfer, ACK remains the standard because it is fast, cheap, gentle enough, and reproducible when the timing is respected.