Ammonium chloride potassium lysis buffer (Report on principles of ACK lysis buffer)

這份報告主要是在大四實習,臨床血清免疫學實驗時老師給的一個報告問題:為什麼ammonium chloride/potassium (A/C-K, hence ACK) lysis buffer可lyse RBC
主要是做peripheral blood lymphocyte immunophenotyping by fluorescence activated cell sorting的實驗
在human peripheral blood中,RBC佔了極多數,若不將其lysis,會有細胞數過多,interference from hemoglobin等的問題。
有許多種方式可打破細胞,包括0.84% NH4Cl, 0.75% CH3COOH, ascorbic acid, saponin或商業化BD, santa cruz, AbDserotec,invitrogen等的lysis kit中可能會用的buffer,常用的是formaldehyde, diethylene glycol或會添加界面活性劑等加速lysis。

主要是在網路上與Wintrobe's clinical hematology中引用資料寫出這份報告

在網路其實蠻難找到實際解釋ACK lysis buffer原理的有關資料

就請各位看倌自行判斷。 (字寫得有點小,請見諒)

This report was on the working principle of ACK lysis buffer in clinical immunohematology during the last year of internship in clinical laboratory science at national taiwan university hospital.
The ACK lysis buffer may work in principle of multiple mechanisms under a isotonic condition.
Ammonium chloride itself has been used for isotonic cell lysis.
Use of potassium bicarbonate may assist this cell lysis process.
The increase of bicarbonate may assist the build up of chloride ions inside of erythrocytes through band 3 proteins, which are chloride/bicarbonate transporters expressed on erythrocytes.
NH4+ is freely diffused across the cellular membrane, so the limit step of the process will probably on chloride ion + ammonium ion to ammonium chloride.
The increase of chloride ion contributes to ammonium chloride build up inside the erythrocytes, thus affecting intracellular osmolity
Under a isotonic condition with increased intracellular ammonium chloride concentration would contribute to influx of H2O eventually causing the burst of erythrocytes.

Lately i found a reference about ACK lysis buffer.
Along with it in the removal of erythrocytes from cell suspensions part, are other 3 methods.
hemolytic Gey's solution, Tris-buffered ammonium chloride, and distilled water
FYI, this book is
Rafael Fernandez-Botran and Vaclav Vetvicka, methods in cellular immunology, 2nd ed, 2002

Alternate recipe from Current protocols:
Current protocols in cytometry, Appendix 2A common stock solutions, buffers, and media
Ammonium chloride lysis solution 10x
1.5M NH4Cl
100mM NaHCO3
10mM Na2EDTA
H2O to 900
adjust pH to 7.4
add H2O to 1L
store for 6 months at 4℃
working solution:dilute stock 10 times fresh before use.

note from the authors:never store lysing buffer at <10x concentration, as it forming ammonium carbonate, rendering ineffective.
sodium bicarbonate buffer may be replated by KHCO3, and disodium by tetrasodium EDTA (8.2mM)
some researcher use 10 fold lower concentration of stock solution

ACK lysis buffer ingredient and FACS protocol from NTUH & CLSMB internship

8.29g NH4Cl (0.15M)
1g KHCO3 (10mM)
0.0372g Na2EDTA (0.1mM)
800mL H2O

use 1N HCl to adjust pH to 7.2-7.4
Add 200mL H2O to 1L
0.2µm filter, preserve under 4℃

1.50µL of EDTA blood add antibody mixture
2.add 1mL ice-cold ACK lysis buffer ( buffer volume to blood volume = 20:1)
3.30min room temp incubation
4.centrifugation 5000rpm 1min, remove supernatant
5.add 1mL 1x PBS resuspend cells
6.centrifuge 5000rpm 1min, remove supernatant
7.add 500µL 1% PFA, resuspend cells and filter into falcon tube

Also Reference

Crippen et al., Analysis of Salmonid Leukocytes purified by Hypotonic lysis of erythrocytes, Jounral of aquatic animal health, 13:234-245, 2001
Paul, Hsieh-Fu Tsai,
Jul 3, 2011, 12:53 PM