The overall goal of this experiment, is to detect chromosomal damage induced by exposure of cells to particulate matter, extracted from ambient air. This method can help answer one key question in the field of particulate matter, and that is does particulate matter exposure induce chromosomal damage? The main advantage of this technique, is it allows direct visualization of the damage induced in the genetic material.
After collecting and analyzing particles according to the text protocol, carry out particle exposure by warming complete growth medium, 0.25%trypsin, and PBS in a water bath at 37 degrees celsius, for at least 30 minutes before culture initiation. After washing raw 264.7 cells, add one milliliter of the warm trypsin solution to the dish and dislodge the cells from the bottom of the tissue culture vessel by scraping. Then collect the cells and make a single-cell suspension by repeated pipetting.
With trypan blue, count the number of cells, then use pre-warmed complete medium to plate them at a density of 8000 per square centimeter in a 100 millimeter tissue culture dish. Then use complete medium to adjust the volume to 10 milliliters for a final concentration of 50, 000 cells per milliliter. Incubate the cells at 37 degrees celsius, and 5%CO2 for 24 hours to allow them to attach.
In a bio-safety cabinet, use sterile PBS to dilute filtered particles at a concentration of 0.5 milligrams per milliliter, for a 5 microgram per milliliter treatment dose, and at 5 milligrams per milliliter, for a 50 microgram per milliliter treatment dose. To treat cells with particulate matter, check the cultured cells under a microscope with 10x magnification for cell growth, cell morphology, culture condition, and contamination. The cells should be at least 30%confluent at this stage.
Using a sterile pipette, aseptically aspirate the medium and use two milliliters of 37 degrees celsius PBS to wash the culture vessel two times. Add a pre-determined dose of particulate matter with fresh medium and incubate the cultures at 37 degrees celsius and 5%CO2 to parallel the exposure used for concurrent genotoxic and epigenotoxic analysis. To arrest cells at metaphase, after washing the cells add two milliliters of pre-warmed medium with five microliters per milliliter of colcemid solution to the culture, and incubate at 37 degrees celsius and 5%CO2 for two hours.
After the incubation, completely remove the medium and use warm PBS to wash the cells two times. Then add trypsin solution and incubate the cultures at 37 degrees celsius for one minute. Using a cell scraper, detach the cells and add 10 milliliters of PBS to inactivate the trypsin.
Then pipette the cells up and down at least ten times to create a single-cell suspension, and transfer the cells into a 15 milliliter, conical centrifuge tube. Place the tubes in a swing out centrifuge, and spin the cell suspension at 400 times g and room temperature for five minutes. To carry out hypotonic treatment, remove the supernatant from the tubes without disturbing the cell pellet, and leave approximately 0.5 milliliters of PBS.
Gently break the cell pellet and use a P200 pipette to make a single cell suspension. Then add four milliliters of pre-warmed potassium chloride solution drop wise with gentle shaking. Incubate the cell suspension in a 37 degree celsius water bath for 20 minutes.
After the hypotonic treatment, fix the cells by adding an equal volume of fixative to the tube, and gently mix by inverting the tube. Centrifuge the tubes at 400 times g at room temperature for five minutes and remove the supernatant. This stage, the cell pellet will have increased in size and become whitish in color and more visible.
Remove the supernatant. Add four milliliters of fresh fixative, and incubate the tubes at room temperature for 30 minutes. Then centrifuge the tubes, remove the supernatant, and use four milliliters of fixative to wash the cells two more times.
Immerse glass slides completely in 10%liquid detergent for 30 minutes. Then rinse the slides thoroughly in cold running tap water for 30 minutes. Use distilled water to rinse the slides three times to completely remove the detergent.
Then immerse them in distilled water, and store the slides in the refrigerator overnight. Drop ten microliters of cell suspension on a chilled wet slide, and allow it to dry overnight at room temperature. To stain and mount the samples, prepare 50 milliters of giemsa staining solution by mixing one part of giemsa stain solution, with one part of PBS, and pour into a coplin jar.
Immerse the slides in staining solution for 20 minutes, then rinse the slides in distilled water to remove excess stain, and immediately use a hair dryer to dry the slides. Leave the slides at room temperature overnight. The following morning, apply a single drop of mounting medium on the slide.
Gently place a cover slip on top, and allow the mounting medium to slowly spread over the slide. Then use a paper towel to remove excess medium. Finally, use a bright field microscope at 100x magnification to examine the structural chromosomal aberrations in the metaphase spreads.
This image shows that a normal mouse metaphase spread, will have 40 acentric chromosomes. Treatment with particulate matter induces various chromosomal aberrations, such as a chromatid break, an acentric fragment, a ring chromosome, a dicentric chromosome, double minutes, a Robertsonian translocation, and an endoreduplication. While attempting this procedure, it is important to remember that only proliferating cells should be used.
Following this procedure, other methods, like M-FISH and SKY, can be used to answer additional questions. Like whether inter-chromosomal, stable aberrations are present. Don't forget the cautions and carcinogens, and precautions such as wearing gloves, should always be taking while performing this procedure.
