Extraction and Identification of Micronuclei from Human Peripheral Blood Lymphocytes

Summary

This protocol outlines a method for extracting and purifying micronuclei from human lymphocytes using sucrose density gradient centrifugation. It provides an experimental basis for investigating the composition and function of micronuclei.

Abstract

A micronucleus (MN) is an abnormal nuclear structure that forms in cells, particularly bone marrow or blood cells, when exposed to external damage, such as radiation, due to unresolved DNA damage or mitotic errors. Once formed, MNs can actively contribute to various carcinogenic processes, including inflammatory signaling and chromosomal genetic rearrangements. MNs contain nuclear DNA, histones, nucleoprotein fragments, and other active proteins, which are closely associated with their functions. Studying the formation and components of MNs is crucial for understanding their role in driving carcinogenic processes. The extraction and purification of MNs are essential to achieving these research objectives. However, the instability of the MN nuclear membrane and its susceptibility to rupture make these processes technically challenging. Currently, only a few studies have reported the use of density gradient centrifugation for MN separation. This study summarizes and simplifies the processes of MN separation and purification. Human peripheral blood lymphocytes exposed to radiation were isolated, and MNs were separated and purified using sucrose buffers of different concentrations in a two-step process. The integrity and purity of the MNs were verified, providing a clear and practical demonstration of the experimental procedure for researchers investigating the causes and functions of MNs.

Introduction

The micronucleus (MN) is a subcellular structure in the cytoplasm that contains nuclear DNA, histones, and nucleoprotein fragments, surrounded by membrane structures^1,2. The MN is completely separate from the main nucleus and typically appears near it in an oval or circular shape, with a size ranging from 1/16 to 1/3 of the main nucleus's diameter. The formation of MNs is primarily associated with acentric chromosome fragments, chromosomal missegregation, dicentric chromosome breakage, chromosome instability, and the aggregation of double minutes (DBs)³. MNs rarely occur in healthy ....

Protocol

All experiments involving human peripheral blood samples were conducted in accordance with relevant guidelines and regulations. This study was approved by the Ethical Committee of Nuclear Industry 416 Hospital, China (2020 Review [No. 48]), and informed consent was obtained from all participants. The exclusion criteria included individuals with major chronic diseases, such as tumors, gout, or blood disorders, as well as those exposed to radioactive or genotoxic substances in their occupation. For this study, 10 mL of peripheral blood was collected from a healthy 28-year-old male into a blood collection tube containing heparin sodium as an anticoagulant. The sample was....

Results

After radiation exposure, human peripheral blood was incubated with RPMI-1640 for 60 h, and then Cytochalasin B was added for micronucleus (MN) preparation. Lymphocytes were isolated using a lymphocyte separation solution, followed by lysis with a specially configured cell lysate and gentle homogenization in a glass homogenizer. The homogenate was mixed 1:1 with 1.8 M sucrose buffer. Unpurified micronuclei were obtained after the first sucrose density gradient centrifugation, and purified micronuclei were obtained a.......

Discussion

In this method, irradiated human peripheral blood lymphocytes were used for the isolation and purification of micronuclei (MNs). Many previous studies have reported the detailed steps for cell lysis and primary separation of MNs^4,32,33,34. The cell lysis solution typically includes 0.1% NP-40 to disrupt the cell membrane while having minimal effect on the nuclear membrane, thereby preserving th.......

Materials

Name	Company	Catalog Number	Comments
15 mL conical tube	Thermo	339650
4% paraformaldehyde solution	Beyotime	P0099
50 mL conical tube	Thermo	339652
Anti-γ-H2AX antibody	Bioss	bsm-52163R
Bovine serum albumin	Beyotime	ST023
Calcium chloride	Biosharp	BS249
Cytochalasin B	Macklin	14930-96-2
DAPI dyeing solution	Bioss	S0001
Dithiothreitol (DTT)	Coolaber	CD4941
EDTA	Biosharp	BS107	Ethylene Diamine Tetraacetic Acid
Fluorescence microscope	Nikon	Ti2-U
Homogenizer	Ybscience	YB101103-1(20 mL)
Horizontal controlled temperature centrifuge	Thermo	Sorvall ST1R plus	Brake speed is set to "5"
Human lymphocyte separation solution	Beyotime	C0025
Magnesium acetate	Macklin	M833330
NP-40	Coolaber	SL9320	10% solution
Phosphate buffer solution(PBS)	HyClone	SH30256.01B
Protease inhibitors	Coolaber	SL1086	Cocktail (100×)
Secondary antibody	Bioss	Bs-0295G	Goat Anti-Rabbit IgG H&L/FITC
Spermidine	Coolaber	CS10431
Spermine	Coolaber	CS10441
Sucrose	Coolaber	CS10581
Tris-HCl	Biosharp	BS157
Triton X-100	Beyotime	P0096