We are addressing LPS and ATP-introduced death of PMA-differentiated THP-1 macrophages. LPS and ATP-introduced deaths are not limited to pyroptosis, but apoptosis and necroptosis can also occur. This will add in your better understanding of cell death after LPS and ATP stimulation and choosing more suitable experimental methods.
Chrome technology mainly studies the outer structures in tissues, cells and microorganisms. Chrome provides neo-native structure data for diverse biomolecular complexes. For example, using Chrome techniques to perform 3D reconstruction can help us further understand the characteristic of pyroptotic and necroptotic parts.
Although pyroptosis is considered dominate in LPS and ATP-introduced cell death, one cannot determine the propagation of this death forms or whether they exist. Validating diverse cell death and measuring them will be our area of investigation in the future. To begin, obtain differentiated THP-1 cells and remove the spent medium from the wells of the culture plate.
After washing the cells with PBS, add a serum-free medium containing lipopolysaccharide or LPS to the cells and incubate the plate. Then, add the adenosine triphosphate or ATP stock solution to the model group and incubate the plate for 45 minutes at 37 degrees Celsius with 5%carbon dioxide. Next, aspirate all the supernatant from the wells of the culture plate.
After washing the cells two times with PBS, add 500 microliters of 0.05%trypsin without EDTA to each well and incubate for 30 seconds. To stop cell detachment, add one milliliter of RPMI 164O medium and transfer the cells into a five-milliliter tube. Centrifuge the tube at 300 G for three minutes at room temperature.
After resuspending the cells in one milliliter PBS, place the tube with cells in a water bath at 37 degrees Celsius for three minutes and centrifuge. To resuspend the cells, add 100 microliters of 1X binding buffer and transfer the cells into a new five-milliliter tube. Incubate the control and model tubes with appropriate reagents in the dark at room temperature.
To terminate the incubation, add 400 microliters of PBS to the tube and vortex it gently. Filter the cell suspension in a 35-micrometer nylon mesh, fixed to a five-milliliter polystyrene round bottom tube. Dip a clean cover glass in chromium alum solution for two hours and dry it in a 37 degrees Celsius oven.
Pellet the trypsinized cells as demonstrated earlier, and fix the cells with 500-microliters of electron microscope fixative for two hours at room temperature, followed by overnight incubation at four degrees Celsius. After collecting the cells from the fixed solution, pellet them at 300 G for three minutes at room temperature. Add 100 microliters of PBS and gently blow away the cells.
Drop the cell suspension onto a cover glass and let it stand for five minutes. Aspirate all the supernatant and wash the cells three times with PBS for five minutes each. Add 500 microliters of 1%osmic acid fixation solution.
After one hour, aspirate all the supernatant. After three PBS washes, dehydrate the samples in increasing concentrations of ethanol. Switch on the critical point dryer.
Open the carbon dioxide tank and cool the chamber to 10 degrees Celsius. Quickly wrap the sample with filter paper and put it into the chamber when the chamber pressure is zero pounds per square inch. Once the temperature stabilizes to 35 degrees Celsius and pressure reaches 1, 250 pounds per square inch, depressurize at 100 pounds per square inch per minute.
Take out the sample when the chamber pressure is zero. Finally, using conductive tape, mount the specimens on scanning electron microscope aluminum specimen holders. Employing a sputter coater, cover the samples with a two to five-nanometer layer of gold.
Flow cytometry analysis showed that after LPS/ATP stimulation, the cells in the QT region increased significantly, indicating cell death. Scanning electron micrographs of the plasma membrane surface showed characteristics of pyroptosis, including membrane blebbing and rupture in LPS/ATP stimulated cells, while the control cells looked normal. Additionally, LPS/ATP stimulation led to the appearance of apoptotic characteristics in the cells, such as membrane blebbing and cell shrinkage, and cells with necroptosis characteristics including cell swelling and ruptured membrane were observed.
