The overall goal of this procedure is to illustrate an easy to use ex vivo model to investigate fresh, carotid or coronary artery plaques. This is accomplished by obtaining fresh plaques from patients undergoing endarterectomy or coronary artery bypass. The second step is to cut the specimen into small pieces.
Next is the random distribution of plaque pieces into a 48 well plate containing RPMI medium. The final step is adding a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. Ultimately, RNA isolation QPCR, immunohistochemistry and EISA can be used to analyze the plaque culture experiment.
The method introduced here can help answer key questions in the vascular inflammation field, such as increasing our knowledge on the pathophysiology of human atherosclerosis. The implications of this technique extend toward diagnosis and therapy of atherosclerosis cause. This is the only method to study the role of interesting molecules on the inflammatory milieu inside human atherosclerotic lesions.
By this, it may allow the identification of novel therapeutic targets and development of novel therapies demonstrating the procedure will be done by Nadine Baskins, an excellent technician from a laboratory. Begin this procedure by adding RPMI medium to a cell culture dish. Place the plaque tissue in the dish such that it is completely covered with medium.
Then hold the edge of the plaque tissue carefully with sterile forceps. Subsequently, divide the plaque tissue in half. Cut off the edge of the plaque sample using a sterile scalpel.
Assess the lesion morphology macroscopically and check if it is calcified lipid rich, ruptured, or has any thrombus or fibrosis. Discard the plaques with severe calcification or fibrosis. Next, cut the plaque tissue into homologous small pieces.
Shock Freeze two plaque pieces for controls and store them in liquid nitrogen until use. Then prepare a 48 well plate by adding 500 microliters of RPMI medium to each well for stimulation. Add one microgram per milliliter of lipopolysaccharide to the well.
After that, randomly plant two plaque pieces in the wells for each group, and use unstimulated plaque pieces as controls. Next, add the substance of interest, such as specific cytokines or chemokines. Then culture the plaque pieces for three hours, eight hours, or 24 hours at 37 degrees Celsius in humidified air containing 5%carbon dioxide.
In this procedure, harvest the plaque tissue pieces after culturing for the indicated time. Then shock. Freeze the plaque pieces for mRNA isolation and CD NA synthesis.
Afterward, collect the supernatant and store it at minus 20 degrees Celsius. For SA analysis to extract RNA from the cultured plaque pieces, use tissue lyer for homogenization. Next, isolate the RNA using the kit.
Then place the samples in the spectrophotometer to determine the RNA quantity and quality. And use the Barringer CDNA kit for reverse transcription. For quantitative PCR.
Use the Roche real-time PCR kit with cyber green. In this experiment, the atherosclerotic plaque pieces were immediately shock, frozen or cultured for eight or 24 hours. And the expression of indicated cytokines was analyzed by Q-R-T-P-C-R.
The results shown here represent the average of five independent experiments and five atherosclerotic lesion pieces for each group were used for the indicated time points. This figure shows the LPS stimulation of cultured plaque pieces induced an increase of various inflammatory molecules. Here are the representative Eliza measurements of different cytokines in the supernatant of plaque pieces treated with LPS or on stimulated for eight hours.
And here is the representative western blotting for IRC one two and phosphorylated IRC one two In the LPS stimulated and control cultured atherosclerotic lesions after three hours, Once mastered, the preparation of the plaque can be done in one hour to initiate the tissue culturing. Following this procedure, various methods like quantitative PCR, Western blotting flow cytometry or immunohistochemistry can be performed in order to analyze the results of the x vivo experiment. This technique passed the way for researchers in the field of atherosclerosis to directly explore the inflammatory failure in human atherosclerotic lesions.
