The scope of our research involves developing and validating a noninvasive method to measure carbon content in airway macrophages as a biomarker for exposure to carbon-containing particulate matter. We aim to determine the accuracy and the reliability of this method of large-scale individual exposure assessments. Our protocol provides a non-invasive, precise, standardized method to measure carbon content in airway macrophages.
As an internal exposure biomarker, this is an effectively and accurately individual exposure to carbon-containing particulate matter. Our protocol offers a direct reliable biomarker for individual exposure to carbon-containing particulate matter. This method allows for large-scale quantification using induced sputum, enhancing both accuracy and reliability compared to other techniques.
Begin by collecting a two milliliter sputum sample in a centrifuge tube. Then, add 20 to 30 milliliters of the prepared fixative solution to the tube and mix the contents thoroughly. To prepare the digestive solution for cell suspension, dissolve 0.1 grams of dithiothreitol in 100 milliliters of saline, according to the required dosage.
Now, centrifuge the tube containing the sputum at 1, 998 G for 30 minutes at four degrees Celsius. Then, discard the supernatant. Add an equal volume of sputum digest to the precipitate.
Vortex and shake the mixture thoroughly. Afterward, place the tube in a 37 degrees Celsius water bath until complete liquefaction. Then, filter the liquified mixture using a 70 micrometer filter membrane.
Centrifuge the filtered solution at 500 G for seven minutes at four degrees Celsius. Discard the supernatant, keeping the cell precipitate. Now, resuspend the cell precipitate in Duchenne phosphate buffer.
Centrifuge the solution at 500 G for seven minutes at four degrees Celsius to obtain a pure cell precipitate. For cell smear preparation, add 200 to 600 microliters of phosphate buffer to the cell sediment and mix thoroughly. Then, take 20 microliters of the cell suspension and create a cell smear.
Fix the dried smear with a commercially available staining solution for 10 seconds. Afterward, immerse the smear in staining solution A for eight seconds. During staining, gently lift the slide up and down, then rinse off excess stain with running water.
Small aggregates of distinctly visible black carbon particles were observed within the cells, varying in the amount of carbon particles. To begin, image the stained sputum slides under a microscope. Randomly select well-stained and morphologically intact macrophages and capture their images.
Using the ImageJ software, measure the scale for accurate pixel conversion. Select Measure and click Set Scale. Enter the Distance in pixels, actual length, and the Unit of length, micrometers.
Then, tick the Global option. Now, outline the cells with an irregular shape and remove the background using freehand selections. Under Edit, select Clear Outside.
Measure the total area of the cells through Analyze and Measure. Cut out the nucleus with Edit and Cut. To convert the gray scale image to black and white.
Click Image, Type, and 8-bit. To adjust the gray scale value based on cell staining to count carbon particles accurately, go to Image, choose Adjust, Threshold, and Apply. Finally, click Analyze and select Measurement.
Carbon black packaging workers had a higher carbon content in airway macrophages than the control group.
