ACK Red Blood Cell Lysis Buffer

Dillon Corvino

Standard ammonium–chloride–potassium (ACK) lysis buffer for selective removal of red blood cells from murine or human single-cell suspensions.
Published

September 18, 2026

Protocol ID: BUF-ACK-LYSIS Version: v1.2 Author: Dillon Corvino Date: September 18, 2026

Purpose

ACK lysis buffer is used to selectively lyse erythrocytes in single-cell suspensions derived from mouse or human tissues (e.g. liver digests, spleen, PBMC preparations). Under brief incubation, the buffer efficiently removes red blood cells while preserving leukocyte viability and integrity.

Linked protocols

  • MUS-IEL-ISO
  • MUS-SPL-DIS
  • MUS-LNG-DIG
  • MUS-BM-ISO
  • MUS-BLD-PRC
  • MUS-TMR-DIG
  • MUS-LIV-DIG

Working buffer composition

Component Final concentration Notes
Ammonium chloride (NH4Cl) X mM Primary erythrocyte-lysing component
Potassium bicarbonate (KHCO3) X mM Buffering capacity
EDTA X mM Chelates divalent cations; reduces cell clumping
Sterile distilled water — Solvent
pH 7.2 Adjusted after dissolution
Note

This formulation reflects the standard ACK buffer used in immunology workflows, with explicit pH adjustment to ensure reproducibility across batches.

Preparation

A. Preparation of stock (if applicable)

Not applicable — prepared directly as the working buffer.

B. Preparation of working buffer

Preparation of 1 L ACK lysis buffer

  1. Use a clean 1 L glass bottle or beaker.
  2. Add the following dry reagents:
    • 8.26 g NH4Cl
    • 1.00 g KHCO3
    • 0.037 g (37 mg) EDTA
  3. Add 1 L sterile distilled (bidest) water.
  4. Mix thoroughly by stirring or gentle swirling until all components are fully dissolved.
  5. Measure pH and adjust to pH 7.2 using dilute HCl.
  6. Sterile-filter the solution using a 0.22 um vacuum filter unit.
  7. Aliquot if desired under sterile conditions.

Labelling

Clearly label each bottle or aliquot with: - Buffer name: ACK Lysis Buffer - Date of preparation - Preparer initials - Sterile - pH adjusted to 7.2

Storage and stability

  • Store at 4 °C.
  • Stable for up to 3 months when sterile.
  • Do not freeze, as freezing may alter buffer performance.

Reagent details

Component Supplier Cat# Notes
NH4Cl [TBD] [TBD] Powder; analytical or cell-culture grade
KHCO3 [TBD] [TBD] Powder; hygroscopic — store tightly sealed
EDTA [TBD] [TBD] Powder; cell-culture grade
Distilled water [TBD] [TBD] Sterile (bidest)
HCl [TBD] [TBD] For pH adjustment

Safety (brief)

  • Wear gloves and eye protection; ammonium chloride is an irritant.
  • Dispose of unused buffer and erythrocyte-containing waste according to biological/chemical waste regulations.
  • For use in primary tissue processing, preparation and sterile filtration should be performed using appropriate aseptic technique.

Version history

Version Date Author Changes
v1.0 2025-11-21 Dillon Corvino Initial Quarto buffer document for ACK lysis buffer
v1.1 2026-01-09 Dillon Corvino Corrected concentrations, explicit pH adjustment, clarified sterility and labelling requirements
v1.2 2026-09-18 Dillon Corvino Migrated to shared summary:/helper architecture (buffer/protocol redesign).