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Cell permeabilization and stabilization reagent and method of use

By using cell permeabilization reagents with components such as N-acyl sarcosine, the problems of morphological change and antigenic site destruction during the cell permeabilization process in the existing technology are solved, and stable permeability of the cell membrane and antigenic site destruction are achieved. Reserved and suitable for flow cytometric analysis.

Inactive Publication Date: 2008-01-30
BECKMAN COULTER INC
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  • Summary
  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

These reagents permeabilize cell membranes, however they generally do not stabilize cell morphology

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1: Permeabilization reagent composition

[0066] Composition A

[0067] [055] The following permeabilization and stabilization reagent compositions were prepared.

[0068] N-Lauroyl Sarcosine

[0069][056] More specifically, a stock solution of N-lauroyl sarcosine was first prepared. 1.0 g of N-lauroyl sarcosine (Fluka) was pre-dissolved in 1.5 ml of ethanol (96%). 180 μl of pyrrolidine (Aldrich Company) was added to 95 ml of deionized water. The N-lauroyl sarcosine / ethanol solution was then added to the pyrrolidine solution; the pH was adjusted to 5.6 with pyrrolidine or HCl, and the volume was adjusted to 100 ml with deionized water to form a stock solution. The total reagent volume was adjusted to 100 ml with deionized water. The permeabilization and stabilization reagent composition was prepared by diluting 6.25 ml of the stock solution to 100 ml with deionized water and adjusting the pH to 5.3 with pyrrolidine or HCl. The conductivity of ...

Embodiment 2

[0078] Example 2: Effects of pH, Surfactants, and Combinations of pH and Surfactants on Sedimentation of Different Components of Erythrocytes

[0079] [061] Some blood treated with 0.7 mM ethylenediaminetetraacetic acid (EDTA) as an anticoagulant was used for serum preparation. Additional EDTA-treated blood was washed with phosphate-buffered saline (PBS) and diluted with 9 volumes of water to obtain a cell lysate. The 2000g cell lysate was centrifuged for 15 minutes to separate the soluble cell fraction and cell membrane fraction. The particles containing the cell membrane fraction are mixed with a volume of water, the mixture contains 5% by volume of the soluble cell fraction.

[0080] After mixing with composition C and following modified reagent in embodiment 1, monitor the precipitation situation of serum, soluble cell fraction, cell membrane fraction and the bovine serum albumin as contrast:

[0081] 1. Modification reagent 1: Composition C in Example 1 without adding s...

Embodiment 3

[0093] Example 3: Effect of pH, Surfactants, and Combinations of Acidic pH and Surfactants on Antibody Penetration into Erythrocytes

[0094] [067] 0.01 ml whole blood was mixed with 100 [mu]l saline solution and then mixed with 2 ml of each permeabilization reagent variant as described in Example 2. After incubation for 5 minutes, 50 μl of each mixture was added to 50 μl of PBS solution containing 0.2% (w / v) bovine serum albumin and monoclonal antibody conjugated to FITC and anti-α-tubulin ( Beckman Coulter, Miami, USA). α-Tubulin is a molecule that is expressed only in cells. After 15 minutes of incubation, the mixture was mixed with 1 ml of PBS containing 0.5% formaldehyde to stop the reaction and fix the cells to form a sample mixture for analysis.

[0095] [068] The sample mixture was analyzed on an XL flow cytometer (Beckman Coulter, Miami, USA). Cell integrity was analyzed by forward scatter and side scatter. The permeability of the cells was analyzed fluorescently ...

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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.

Description

technical field [0001] [001] The present invention relates to a cell permeabilization and stabilization reagent and method of use thereof for preparing a cell-containing sample for analysis of cellular components. Background technique [0002] [002] It is an urgent requirement to analyze the internal components of cells at the molecular level. Several probes and antibodies have recently appeared, which are generally available for routine research and indicate intracellular structures. These probes and antibodies, because of their macromolecular nature, cannot permeate cells through cell membranes by themselves. Cells therefore need to be treated in order to render the cell membrane permeable (permeabilization grade). This treatment causes a major modification of the outer lipid membrane and, depending on the method used, can lead to loss of cell morphology, or even to the death of the entire cell. [0003] [003] Standard permeabilization methods involve treating cells by ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N37/18A61K38/00
CPCA61K31/198Y10T436/107497
Owner BECKMAN COULTER INC
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