Blood Flowcytometry of CD44+/CD24-, RAD6 and DDB2 in Ovarian Cancer Patients
Description
The data was raw and analyzed data of the blood flow cytometry expression of CD44+/CD24-, RAD6 and DDB2 in chemosensitive and chemosensitive ovarian cancer patients.
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Blood was taken for a flow cytometry test after 6 months of post-chemotherapy observation was completed. if there are patients who experience chemoresistance before the observation period is over, blood will be taken directly at that time for flow cytometry examination before the 6-month observation period is completed. Blood was taken from peripheral blood at five mL and then centrifugated. The supernatant was discarded, and 50 µL was left, after which the cell mixture was resuspended. The markers identified expression CD44+/CD24-, RAD6, and DDB2. The samples were reacted with fluorescent-labeled antibody against CD44+/CD24- (monoclonal anti-human), CD44+ was labeled as PerCP, CD24- was labeled as APC, RAD6 was labeled as PE, and DDB2 was labeled FITC. The four reagents were then removed for leukocytes with CD45 labeled pacific blue. The samples in the Falcon tube were then added with 2.5 µL of CD44 marker, 2.5 µL of CD24 marker, and 2.5 µL of RAD6 and DDB2. Next, they were incubated for 15 min in the dark at room temperature. After incubation, the cells were lysed by using 300 µL of lysing solution, then set again for 15 min in a dark room and at room temperature. Next, 1 mL of FACS flow solution was added and centrifuged at 500 g for 5 min. The supernatant formed was then discarded, added with 500 µL perm wash buffer and centrifuged at 500 g for 5 min; the supernatant created was discarded. To be more optimal, 1 mL perm wash buffer was added again and centrifuged at 500 g for 5 min. The last step was to add 200 µL of 1% paraformaldehyde in phosphate-buffered saline (PBS). After that, the analysis was conducted by using a flow cytometer using four fluorochrome colors. Next, cell identification was conducted by using an automated flow cytometer (BD Facs Calibur). CSCs were then identified through the positive expression of CD44+/CD24- markers and RAD6 and DDB2 were conducted through a positive expression of RAD6 and DDB2 markers with four different colors. Protein percentage is the percentage of expression of protein markers CSCs (CD44+/CD24-), RAD6, and DDB2 in the blood.