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Short answer: A red cell lysis buffer recipe, often called ACK buffer, combines ammonium chloride, potassium bicarbonate and disodium EDTA in distilled water, adjusted to pH 7.2-7.4. Ammonium chloride lyses red blood cells with minimal effect on leukocytes. Ready-made 1X and 10X versions exist for labs that prefer not to mix their own.
Definition and scope
Red cell lysis buffer (also written RBC lysis buffer or erythrocyte lysis buffer) is a research reagent used to remove red blood cells from a sample so that white cells can be counted, stained or analysed. It is typically used on whole blood, or on tissue preparations such as spleen and bone marrow. Thermo Fisher states that its RBC lysis buffers contain ammonium chloride, which lyses red cells with minimal effect on leukocytes. The same manufacturer labels the product for research use only, not for use in diagnostic procedures.
This page covers what the buffer contains, how the main routes to it differ and where it matters for lab sourcing. It is not a bench protocol: exact concentrations, volumes and timings should be taken from the validated protocol or the manufacturer’s instructions for the sample type in use.
Components and pH of the home-made ACK buffer
AAT Bioquest lists three components for its ACK lysis buffer recipe, with the molecular weights used for weighing out each salt:
- Ammonium chloride (MW 53.49), the lysing agent.
- Potassium bicarbonate (MW 100.12).
- Disodium EDTA (MW 372.24).
The components are dissolved in distilled water, the pH is adjusted to 7.2-7.4, and the volume is made up to 1 L. AAT Bioquest gives storage of up to 6 months at room temperature. Published recipes differ in the exact concentrations they use, so treat the component list and pH window as the common frame and take quantities from the protocol your lab has validated.
Home-made, ready-made 1X/10X and one-step fix/lyse compared
The table separates three routes by what the manufacturer and recipe sources named at the end of this page state.
| Route | What is stated | Source |
|---|---|---|
| Home-made ACK buffer | Ammonium chloride, potassium bicarbonate and disodium EDTA; pH 7.2-7.4; made to 1 L in distilled water; stored up to 6 months at room temperature | AAT Bioquest |
| Commercial 1X or 10X RBC lysis buffer | Contains ammonium chloride; 10X must be diluted 1:10 with room-temperature, reagent-grade water before use | Thermo Fisher |
| One-step fix/lyse solution (10X) | Combines fixation and lysis in one reagent; manufacturer example incubation of 15-60 minutes; composition not stated | Thermo Fisher |
The difference that matters most in practice is the third row: a fix/lyse reagent fixes cells as it lyses them, so it is a different tool from a plain ACK buffer and is not interchangeable with it.
Sample type changes the manufacturer’s example times
Thermo Fisher’s published examples for its RBC lysis buffer show how much the sample matters. For bulk lysis of human whole blood it gives 10-15 minutes at room temperature, with “no more than 15 minutes”. For mouse or rat spleen or bone marrow it gives 4-5 minutes. For antibody staining followed by lysis of whole blood it gives 10-15 minutes for human and 4-10 minutes for mouse. These are one manufacturer’s examples, not universal settings; follow the instructions for your own sample type.
Where it matters for labs and supply decisions
A short checklist drawn from the manufacturer and recipe sources listed below:
- Know which reagent you are buying. A plain RBC lysis buffer and a fix/lyse solution are different products with different incubation ranges.
- Check the concentration on the label. A 10X product needs a 1:10 dilution with reagent-grade water; a 1X product does not.
- Match the manufacturer’s example to your sample. Human blood and rodent spleen carry different times in the same instructions.
- Respect the research-use-only labelling. Thermo Fisher states its lysis buffers are not for use in diagnostic procedures.
- Mind the time ceiling. The manufacturer’s whole-blood example says no more than 15 minutes at room temperature.
Lysis is usually one step in a longer workflow. For what follows it, see the guides on the hemocytometer and the automated cell counter, and on nucleic-acid work, DNA extraction protocol basics. Related background includes fetal bovine serum and the DMEM vs RPMI comparison for culture of the recovered cells.
Common misunderstandings
- “ACK and fix/lyse are the same thing.” They are not. ACK-type buffers lyse red cells; a fix/lyse solution also fixes the remaining cells.
- “The buffer is for diagnostic use.” The manufacturer labels it for research use only.
- “One recipe fits every sample.” Even one manufacturer’s examples change the incubation time between human blood and rodent tissue.
- “Different names mean different reagents.” RBC lysis buffer, red cell lysis buffer and erythrocyte lysis buffer are used for the same type of reagent; the ACK recipe is the common home-made version.
FAQ
What is in an RBC lysis buffer recipe (or erythrocyte lysis buffer recipe)?
The ACK recipe uses ammonium chloride, potassium bicarbonate and disodium EDTA in distilled water at pH 7.2-7.4 (AAT Bioquest). Commercial buffers state that they contain ammonium chloride (Thermo Fisher).
What is an erythrocyte lysis buffer protocol, or an RBC lysis buffer protocol?
It is the validated written procedure a lab follows to lyse red cells in a sample using a lysis buffer. This page does not give a bench protocol: take volumes and times from the protocol or manufacturer instructions for your sample. Thermo Fisher’s published examples range from 4-5 minutes for mouse or rat tissue to 10-15 minutes for human blood.
How long can red blood cells stay in lysis buffer?
Thermo Fisher’s human whole-blood example gives 10-15 minutes at room temperature and says no more than 15 minutes.
Do I need to dilute a 10X lysis buffer?
Yes. For its 10X RBC lysis buffer, Thermo Fisher states the buffer must be diluted 1:10 with room-temperature, reagent-grade water.
How long does home-made ACK buffer keep?
AAT Bioquest gives up to 6 months at room temperature.
Sources
- Thermo Fisher Scientific, Gibco cell culture basics, red blood cell lysis using ACK lysing buffer: https://www.thermofisher.com/au/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/red-blood-cell-lysis-using-ack-lysing-buffer.html (checked 2026-10-29).
- AAT Bioquest, ACK Lysis Buffer recipe: https://www.aatbio.com/resources/buffer-preparations-and-recipes/ack-lysis-buffer (checked 2026-10-29).
