Measuring Carbon Content in Airway Macrophages Exposed to Carbon-Containing Particulate Matters

Summary

This article describes a detailed experimental protocol for measuring the carbon content of airway macrophages with the aim of assessing the internal exposure dose at the level of individual particulate matter exposure.

Abstract

Pulmonary macrophages exhibit a dose-dependent pattern in phagocytizing particles. Following engulfment, these macrophages are subsequently excreted with sputum, rendering macrophages and particles visible and quantifiable under light microscopy. Notably, elemental carbon within the mammalian body originates exclusively from external contaminants. Consequently, the carbon content in airway macrophages (CCAM) serves as a valid exposure biomarker, accurately estimating individual exposure to carbon-containing particulate matter (PM). This article delineates a protocol involving sputum collection, preservation, processing, slide preparation, and staining, as well as macrophage photo acquisition and analysis. After removing the macrophage nuclei, the proportion of cytoplasm area occupied by carbon particles (PCOC) was calculated to quantify carbon content in each macrophage. The results indicate an elevation in CCAM levels after exposure to carbon-containing PM. In summary, this non-invasive, precise, reliable, and standardized method enables the direct measurement of carbon particles within target cells and is utilized for large-scale quantification of individual CCAM through induced sputum.

Introduction

Ambient air pollution is associated with deaths due to respiratory and cardiovascular diseases, posing a serious threat to human health^1,2. Epidemiological data indicate that chronic exposure to ambient particulate matter less than or equal to 2.5 µm in diameter (PM2.5) is responsible for the premature deaths of between 4 and 9 million people globally. PM2.5 was ranked as the fifth most important risk factor for global mortality in the 2015 Global Burden of Disease, Injuries, and Risk Factors Study (GBD)^3,4,5

Protocol

The study received approval from the Medical Ethics Committee of the Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention (NIOHP201604), with written informed consent obtained from all subjects prior to the study and biological sample collection. For this study, carbon black packers who had been working in a carbon black factory for more than 1 year and were exposed to carbon black aerosols were selected. Waterworks workers in a waterworks factory with no significant occu.......

Discussion

This study presents a detailed experimental protocol for using induced sputum-derived CCAM as a biological marker for internal exposure to atmospheric particulate matter. CCAM can be detected and quantified through optical microscopy, serving as a precise internal exposure biomarker reflecting the relationship with health effects. Therefore, there is a need to establish and optimize a convenient, reliable, and efficient induced sputum preservation method and a CCAM quantification method to standardize the methodological .......

Materials

Name	Company	Catalog Number	Comments
3 mL sterile straws	Shanghai YEASEN Biotechnology Co., LTD	84202ES03
50 mL centrifuge tube	Thermo Fisher Scientific, USA	339652
Absolute ethyl alcohol	Sinopharm Group Chemical Reagent Co. LTD	64-17-5
Cedar oil	Shanghai McLean Biochemical Technology Co., LTD	C805296
Diff-quick staining solution	Shanghai YEASEN Biotechnology Co., LTD	40748ES76
Dithiothreitol	Solebo Bio Co., LTD	D8220
Duchenne phosphate buffer (DPBS)	Thermo Fisher Scientific, USA	14190144
Microscope camera	Olympus Corporation of Japan	DP72
Neutral tree gum	Solebo Bio Co., LTD	G8590
Nylon filter membrane 70um	BD Falcon Bioscience, USA	211755
Optical microscope	Olympus Corporation of Japan	BX60
Polyethylene glycol	Sinopharm Group Chemical Reagent Co. LTD	25322-68-3
Ultracentrifuge	Thermo Fisher Scientific, USA	SL40R
Viscous slide	Jiangsu SHitAI EXPERIMENTAL Equipment Co. LTD	188105