Human peripheral blood neutrophil isolation method

All patients who participated in this study signed an informed consent form, and this study was approved by the Ethics Committee of The Third Affiliated Hospital of Southern Medical University (No. 2022-Lunshen-005), and was conducted in accordance with the Declaration of Helsinki (as revised in 2013).

After preparing the disposable venous blood sample collection needles, rubber gloves, disinfectant, and other blood collection equipment, we selected the more vigorous vein in the upper arm of the volunteer and disinfected the blood collection point and its surroundings. The blood was collected in a relatively sterile environment with a lancet into a blood sample collection container containing anticoagulant components, and 10 mL of venous peripheral blood was drawn from each healthy volunteer. The blood collection process was completed with the assistance of nurses from the Department of Rheumatology and Immunology at the Third Affiliated Hospital of Southern Medical University.

Next, we sterilized the blood collection tube and moved it under the ultra-clean table, and added it to a pre-prepared 15 mL centrifuge tube containing a neutrophil separation liquid. We added the blood to the neutrophil separation liquid slowly to stratify them as much as possible. Following centrifugation (500 g for 35 minutes at room temperature), two layers of ring-shaped white cell bands were visible to the naked eye. After extracting the neutrophils in the lower layer, the RPMI-1640 culture medium was used to wash off the excess neutrophil separation solution, and the supernatant was discarded. We then added the RPMI-1640 culture medium to resuspend the cells, added an appropriate amount of red blood cell lysate, and it let stand for 10 minutes at 4 degrees. We then observed the cell pellet after 1,000 revolutions and centrifugation for 5 minutes. If the red blood cells did not appear red, they were considered fully lysed; otherwise, an appropriate amount of red blood cell lysate was added and lysed again until the red blood cells were not visible to the naked eye in the cell pellet after centrifugation. We subsequently discarded the supernatant and washed the cells using the RPMI-1640 culture medium.

Next, we added 1 mL of RPMI-1640 culture medium to resuspend the cells and used a pipette to take 10 µL of the cell suspension and place it in the space between the cell counting plate and the cover glass. We then counted the number of cells in the four large squares on one side of the cytometer under an optical microscope [neutrophil count = (total number of cells in the four large squares/4) × 104 × dilution factor]. Subsequently, the RPMI-1640 culture medium was used to dilute the cells according to the experimental requirements and then set them aside.