Cell permeabilization and stabilization reagent and method of use

• US 7,678,578 B2
• Filed: 02/07/2005
• Issued: 03/16/2010
• Est. Priority Date: 02/07/2005
• Status: Active Grant

• Current Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Original Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Inventors: Rabellino, Enrique, Van Agthoven, Andreas, Malergue, Fabrice
• Primary Examiner(s): Gupta; Anish
• Assistant Examiner(s): Niebauer; Ronald T
• Application Number:
  US11/052,269
• Publication Number:
  US 20060178294A1
• Time in Patent Office: 1,863 Days
• Field of Search: None
• US Class Current: 436/66
• CPC Class Codes: A61K 31/198 Alpha-amino acids, e.g. alanine or edetic acid [EDTA] betaine A61K31/205; proline A61K31/401; tryptophan A61K31/405; histidine A61K31/4172; peptides not degraded to individual amino acids A61K38/00
• CPC Class Codes: Y10T 436/107497 Preparation composition [e.g., lysing or precipitation, etc.]

Abstract

A cell permeabilization and stabilization reagent and method of use are disclosed. The reagent contains a N-acyl sarcosine or a salt thereof, a pH adjusting agent to adjust pH of the reagent in a range from about 4 to about 6; and an aqueous medium; the reagent having a low ionic strength defined by a conductivity of less than 9.0 mS/cm. The reagent further contains bovine serum albumin and glycerol. The reagent may further include an alkyl sulfate surfactant. Upon incubating the cells with the reagent, the reagent permeates the cellular membrane to allow penetration of an intracellular marker, causes intracellular protein aggregation within the cellular membrane, while preserves a cellular constituent for binding with a cellular marker for subsequent analysis by flow cytometry.

Claims

1. A cell permeabilization and stabilization reagent comprising an aqueous solution of:

(a) N-acyl sarcosine or a salt thereof represented by the following molecular structure;

R₁—

CO—

N(CH₃)CH₂COOX₁

wherein R₁ is an alkyl or alkylene group having 8 to 18 carbon atoms, and X₁ is H, Na⁺, or K⁺;

(b) bovine serum albumin; and

(c) a saccharide or glycerol;

said reagent having a pH less than 7 and having a low ionic strength defined by a conductivity of less than 1.2 mS/cm.

2. The reagent of claim 1, wherein said pH is in a range from about 4 to about 6.

3. The reagent of claim 1, wherein said N-acyl sarcosine is N-lauroyl sarcosine.

4. The reagent of claim 3, wherein said N-lauroyl sarcosine is in a concentration range from about 0.1 mM to about 10 mM.

5. The reagent of claim 1, wherein said saccharide is disaccharide, or monosaccharide.

6. A sample mixture composition comprising:

(a) a whole blood; and

(b) a cell permeabilization and stabilization reagent comprising an aqueous solution of 0.1 mM to 10 mM of N-acyl sarcosine or a salt thereof represented by the following molecular structure;

R₁—

CO—

N(CH₃)CH₂COOX₁

wherein R1 is an alkyl or alkylene group having 8 to 18 carbon atoms, and X₁ is H, Na⁺, or K⁺;

said reagent having a pH between 4 and 6 and a conductivity less than 1.2 mS/cm; and

wherein the cellular membrane of blood cells in said whole blood is permeated and intracellular proteins of said blood cells are caused to aggregate by said reagent, while said blood cells are preserved for cellular analysis.

7. The sample mixture composition of claim 6 further comprising a monoclonal antibody.

8. The sample mixture composition of claim 7 further comprising a fixative.

9. A cell permeabilization and stabilization reagent comprising an aqueous solution of:

(a) 0.1 mM to 10 mM of N-acyl sarcosine or a salt thereof represented by the following molecular structure;

R₁—

CO—

N(CH₃)CH₂COOX₁

wherein R1 is an alkyl or alkylene group having 8 to 18 carbon atoms, and X₁ is H, Na⁺, or K⁺; and

(b) saccharide or glycerol;

said reagent having a pH less than 7 and a low ionic strength defined by a conductivity of less than 1.2 mS/cm;

wherein said reagent enables permeation of the cellular membrane and causes aggregation of intracellular proteins of blood cells while preserving said blood cells for cellular analysis.

10. The reagent of claim 9, wherein said pH is in a range from about 4 to about 6.

11. The reagent of claim 9, wherein said N-acyl sarcosine is N-lauroyl sarcosine.

12. The reagent of claim 9, wherein said saccharide is disaccharide, or monosaccharide.