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Flow cytometry detection method for Pig-a gene mutation test in rat body

A technology of flow cytometry and detection method, applied in the field of flow cytometry detection of rat peripheral blood Pig-a gene mutation test, can solve the problems of high cost, increased operation steps, high detection cost, etc., and achieve high sensitivity and stability High reliability, high stability, and cost-saving effect

Active Publication Date: 2020-10-27
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The operation steps involved in the two methods have the disadvantages of complicated operation and high cost: the emission spectrum of the fluorescent dye combination overlaps, which leads to an increase in the method operation steps, and the sensitivity is low; some methods need to destroy or remove white blood cells to eliminate interference, resulting in the operation The steps are cumbersome; the detection costs of the existing two methods are relatively high

Method used

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  • Flow cytometry detection method for Pig-a gene mutation test in rat body
  • Flow cytometry detection method for Pig-a gene mutation test in rat body
  • Flow cytometry detection method for Pig-a gene mutation test in rat body

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] sample collection

[0036] Take 5-week-old male SD rats of SPF grade, weighing 150-160 g. The doses of test substance A were 0, 20, 40, 80, 160mg / kg.bw / d, and the solvent was pure water. The drug was administered orally for 28 consecutive days, and the samples were collected on the 1st, 14th, and 28th day before the test, respectively. Peripheral blood testing.

[0037] When collecting blood, wipe the rat tail with an alcohol cotton ball to fill the blood vessels in the tail, and use a syringe needle to puncture the blood vessels along the parallel direction of the blood vessels. Collect 50 μL of naturally flowing blood into a centrifuge tube (containing 10 μL heparin sodium solution), and mix immediately to prevent coagulation. After the blood collection, the animals should be disinfected and the bleeding should be stopped. The collected peripheral anticoagulated blood was stored in a refrigerator at 4°C and protected from light until testing.

[0038] Reagent prep...

Embodiment 2

[0054] sample collection

[0055] Take 5-week-old male SD rats of SPF grade, weighing 150-160 g. The exposure doses of test substance B were 20, 40 and 80 mg / kg.bw / d, and the solvent was pure water. The drug was administered orally for 3 consecutive days, and peripheral blood was collected on the 28th day of the experiment for testing.

[0056] When collecting blood, wipe the rat tail with an alcohol cotton ball to fill the blood vessels in the tail, and use a syringe needle to puncture the blood vessels along the parallel direction of the blood vessels. Collect 50 μL of naturally flowing blood into a centrifuge tube (containing 10 μL heparin sodium solution), and mix immediately to prevent coagulation. After the blood collection, the animals should be disinfected and the bleeding should be stopped. The collected peripheral anticoagulated blood was stored in a refrigerator at 4°C and protected from light until testing.

[0057] Reagent preparation

[0058] APC mouse anti-...

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Abstract

Aiming at the technical problems in the prior art, the invention provides a rat peripheral blood Pig-a gene mutation test flow cytometry detection method, which comprises the following steps of 1, collecting target tissue cells,2, dyeing a surface antigen,3, dyeing with a nucleic acid dye application solution,and 4, after dyeing is finished, detecting the sample by using a double-laser flow cytometer to obtain data. The detection method distinguishes red blood cells by using forward angular scatter (FSC) and sideangular scatter (SSC), Syto13 labels reticulocyte, CD59-allophycocyanin (APC) labels mutant cells, and the rat peripheral blood Pig-a gene mutation test flow cytometry detection method is simpler, more convenient and economical.

Description

technical field [0001] The invention relates to the technical field of toxicological safety evaluation genotoxicity test, in particular to a flow cytometry detection method for rat peripheral blood Pig-a gene mutation test. Background technique [0002] Pig-a gene mutation test is a genotoxicity test that reflects the end point of gene mutation in vivo established and developed in recent years. It mainly determines the level of mutation after the subject is exposed to mutagenic substances by detecting the expression level of markers on the surface of mutant cells. . In mammals, the Pig-a gene is located on the X chromosome and encodes the N-acetylglucosamine transferase complex, which is mainly involved in the synthesis of glycosylphosphatidylinositol (GPI). When the Pig-a gene is mutated, GPI cannot be synthesized normally, resulting in loss of expression of cell surface proteins (CD59, CD55, etc.) Cytometer for detection. The Pig-a gene mutation test has the advantages ...

Claims

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Application Information

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IPC IPC(8): G01N15/14G01N15/00G01N1/30G01N1/28
CPCG01N15/14G01N15/1434G01N15/00G01N1/30G01N1/28G01N2015/014G01N15/01
Inventor 陈锦瑶霍娇张立实刘运杰马思佳曾珠岳茜岚
Owner SICHUAN UNIV
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