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 cells and are predominantly caused by exposure to exogenous genotoxins, which lead to DNA damage or mitotic errors^4,5. Radiation damage is a significant contributor to MN formation, and human exposure to ionizing radiation (IR) has increased with advancements in clinical and technological applications^6,7. IR exposure induces single-strand breaks (SSBs) and double-strand breaks (DSBs) in cellular DNA, which can result in cell death or apoptosis^8,9,10. MNs are fragments of chromosomes, whole chromosomes, or chromatids produced due to unrepaired or mismatched DSB repair. They serve as critical indicators of the degree of damage caused by IR^7,11,12. A comprehensive understanding of the components within MNs is essential for mitigating the side effects of radiation therapy and minimizing public exposure to unwanted radiation^6,13. However, the proteins and nucleic acids contained within MNs remain incompletely characterized to date.

MNs are believed to result from the induction of various types of DNA damage. Their replication mechanisms and DNA repair abilities are impaired, leading to extensive DNA damage within a short period^3,14,15. MNs are also important indicators of chromosomal instability, which is consistently present in precancerous cells^{3,14,16,17,18,19}. MNs have long been used as biomarkers for genotoxicity, tumor risk, and tumor grade^3,20,21. Once formed, MNs can actively drive numerous carcinogenic processes, including inflammatory signaling^22,23and chromosomal genetic rearrangement^24,25,26. For instance, MNs initiate an inflammatory cascade by recruiting the viral pattern recognition receptor (PRR) cyclic GMP-AMP synthase (cGAS) from the cytoplasm^4,22,23,27. Other proteins that may be present in MNs include exonucleases²⁸, transcriptional mechanisms⁴, and translation cofactors. It remains unclear whether MNs assemble a unique, biased protein profile from the cytoplasmic protein library or passively acquire high-abundance proteins from the nucleus and cytoplasm during their formation. Beyond individual proteins such as cGAS, the extent to which specific environments influence the overall MN composition is not yet determined. The understanding of the entire micronuclei landscape is limited, with no publicly available datasets for exploration. Therefore, extracting complete and purified MNs is urgently needed for in-depth and comprehensive analyses of their components.

One reason for delayed DNA replication in MNs may be replication stress caused by a lack of enzymes and cofactors required for DNA synthesis and repair. This deficiency may result from defective assembly of the MN nuclear envelope, leading to the absence of a nuclear pore complex²⁰. Consequently, MNs are unable to import key proteins essential for maintaining nuclear membrane integrity and genome stability^2,29,30,31. The incomplete MN nuclear membrane is prone to rupture, making MN extraction highly challenging.

Currently, MNs are primarily extracted using sucrose density gradient centrifugation^27,32,33and purified by flow cytometry³⁴. In this study, the separation and purification process of MNs was summarized and simplified. Peripheral blood lymphocytes from humans exposed to radiation were isolated, and MNs were purified twice using sucrose buffers of different concentrations. The integrity and purity of the MNs were verified, providing researchers with a practical and intuitive experimental demonstration for studying the causes and functions of MNs.

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 irradiated in vitro with ⁶⁰Co γ-rays at 37 °C using a gamma air kinetic energy therapeutic-level standard device. The irradiation dose was set to 4.0 Gy, with a dose rate of 0.6350 Gy/min. Following irradiation, the sample was incubated at 37 °C for 2 h, inoculated into RPMI-1640 medium, and cultured at 37 °C for 60 h. Details of the reagents and equipment used in this study are provided in the Table of Materials.

1. Preparation of micronucleus and nucleus

1. Add Cytochalasin B to the peripheral blood cell medium to achieve a final concentration of 10 µg/mL. Incubate the mixture for 45 min at 37 °C.
   NOTE: Cytochalasin B is an actin polymerization inhibitor that facilitates the efficient separation of micronuclei from the nucleus prior to cell lysis³⁵.
2. Centrifuge the blood cells at 200 × g for 5 min at 4 °C.
3. Resuspend the blood cells in 10 mL of 0.01 M PBS pre-chilled at 4 °C.

2. Isolation of lymphocytes

1. Add 15 mL of human lymphocyte separation solution into a 50 mL conical tube. Carefully layer the suspended peripheral blood cells on top of the separation solution.
2. Centrifuge the tube at 400 × g for 30 min at 4 °C.
   NOTE: After centrifugation, the blood cells in the tube will separate into four distinct layers from top to bottom: the first layer is the plasma layer (platelets), the second layer is the ring milky white lymphocyte layer, the third layer is the transparent separation liquid layer, and the fourth layer is the red blood cell layer.
3. Carefully transfer the second layer (lymphocytes) into a separate centrifuge tube. Add PBS (0.01 M) at three times the volume of the transferred cells, mix well, and centrifuge at 200 × g for 10 min at 4 °C. Discard the supernatant using a pipette.

3. Lysis of lymphocytes

1. Place the cell pellet collected in the conical tube on ice.
2. Add 6 mL of cold lysis buffer (Table 1, Supplementary File 1) and resuspend the cell pellet in the lysis buffer.
   NOTE: The lysis buffer should be supplemented with the ingredients listed in Table 2, Supplementary File 1, immediately before use.
3. Transfer the cell lysate to a glass homogenizer.
4. Gently use a loose grind to manually homogenize the sample by moving the homogenizer up and down 10 times.
5. Transfer the homogenized cell lysate back to the 50 mL conical tube.
   NOTE: Perform all steps gently and keep the sample on ice.

4. Initial isolation of MNs via a sucrose density gradient

1. Mix 5 mL of the cell lysate with an equal volume of 1.8 M sucrose buffer (Table 3, Supplementary File 1) to achieve a 1:1 ratio of lysate to sucrose buffer.
   NOTE: The 1.8 M sucrose buffer should be supplemented with the ingredients listed in Table 4, Supplementary File 1, immediately before use.
2. Prepare the sucrose density gradient: Add 15 mL of 1.6 M sucrose buffer (Table 5, Supplementary File 1) to the bottom of a 50 mL conical tube. Slowly add 20 mL of 1.8 M sucrose buffer (Table 3, Supplementary File 1) on top of the 1.6 M sucrose buffer.
   NOTE: Both the 1.6 M sucrose buffer and 1.8 M sucrose buffer should be supplemented with the ingredients in Table 4, Supplementary File 1, respectively, immediately before use. To add the sucrose buffers, place the tip of the pipette against the top inner side of the conical tube and slowly pipette the solution.
3. Slowly pipette 10 mL of the [Lysate 1:1 1.8 M sucrose buffer] mixture on top of the two-layer sucrose density gradient.
   NOTE: Add the layers slowly to ensure that the middle 1.8 M buffer layer does not mix with the lower 1.6 M buffer layer, and the upper 1:1 mixture of lysed cells and 1.8 M sucrose buffer does not mix with the 1.8 M buffer layer.
4. Centrifuge the tube in a horizontal rotor centrifuge at 1000 × g for 20 min at 4 °C.
   NOTE: After centrifugation, the liquid will separate into three layers: the upper layer contains approximately 3 mL of cell debris, the middle layer contains approximately 3 mL of micronuclei (MNs), the lower layer contains approximately 39 mL of nuclei.
5. Remove the upper 3 mL of liquid near the surface and collect the 3 mL of unpurified micronuclei from the middle layer for the second sucrose density gradient.

5. Secondary purification of MNs via a sucrose density gradient

1. Prepare the sucrose density gradient:
   1. Add 3 mL of 1.8 M sucrose buffer (Table 3, Supplementary File 1) to the bottom of a 15 mL conical tube.
   2. Add 3 mL of 1.5 M sucrose buffer (Table 6, Supplementary File 1) on top of the 1.8 M sucrose buffer.
   3. Finally, add 3 mL of 1.4 M sucrose buffer (Table 7, Supplementary File 1) on top of the 1.5 M sucrose buffer.
      NOTE: Position the tip of the pipette against the top inner side of the conical tube and slowly pipette the solution.
2. Slowly pipette 3 mL of the middle layer from the second sucrose density gradient onto the top of the three-layer sucrose density gradient.
   NOTE: Add the layers slowly to avoid mixing.
3. Centrifuge the tube in a horizontal rotor centrifuge at 500 × g for 20 min at 4 °C.
   NOTE: After centrifugation, the upper layer will contain 1-4 mL of purified micronuclei (MN), and the lower layer will mainly contain 8 mL of nuclei.
4. Reserve the upper 1-4 mL of purified micronuclei for later use.

6. Identification of MNs

1. Take the unpurified micronucleus solution (obtained in step 4) and purified micronucleus solution (obtained in step 5) and prepare cell smears on the slide.
2. Add 4% paraformaldehyde solution to fix the sample at room temperature (RT) for 15 min.
3. Wash the slides with PBS (0.01 M) twice, 3 min per wash.
4. Add 0.1% Triton X-100 to permeabilize the cells at RT for 5 min.
5. Block the cells in 5% BSA at RT for 30 min.
6. Add the primary antibody against anti-γ-H2AX (diluted in 5% BSA, 1:200) and incubate at RT for 1 h.
7. Wash the slides with PBS (0.01 M) twice, 5 min per wash.
8. Add the secondary antibody (diluted in 5% BSA, 1:500) and incubate at RT for 1 h in the dark.
9. Add DAPI dye solution and incubate at RT for 15 min.
10. Wash the slides with PBS (0.01 M) twice, 3 min per wash.
11. Apply a sealing solution to the slide to seal it.
12. Observe fluorescence under a fluorescence microscope (magnification: 400×, auto-focus, and auto-exposure), and count the micronuclei using a cell counting plate.
    NOTE: 4% paraformaldehyde, 0.1% Triton X-100, and 5% BSA are diluted with PBS (0.01 M). γ-H2AX (Gamma H2AX) is a phosphorylated form of the Histone H2AX protein, formed in response to DNA damage. In the nucleus, the phosphorylation of Histone H2AX is concentrated in certain regions, so anti-γ-H2AX fluorescence may appear spotty. The phosphorylation of Histone H2AX is concentrated in the micronucleus, resulting in overall brighter fluorescence with distinct spots³⁶. DAPI is a fluorescent dye that binds strongly to DNA and is often used in fluorescence microscopy. Since micronuclei contain broken DNA and histone fragments, they can be stained by anti-γ-H2AX fluorescent antibodies and DAPI. The fluorescence will indicate the number, size, and completeness of the micronuclei, helping distinguish them from the nuclei and assess the purification effectiveness³².

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