Ficoll hypaque plus

Peripheral blood was drawn from healthy donors and collected in heparin blood collection tubes. Human neutrophils were isolated from the peripheral blood using a previously described method (Cheung et al., 2015 (link)). Briefly, peripheral blood was resuspended in RPMI 1640, layered with Ficoll Hypaque Plus (Sigma-Aldrich), and then centrifuged at 1000 g for 20 min. The supernatant was discarded, and the red blood cell (RBC) pellet was incubated with red blood cell lysis buffer (CWBiotech, China) at a 9-fold volume for 15 min at 37°C to remove erythrocytes. After centrifugation at 1000 g for 15 min, the supernatant was aspirated, and the cell pellet was washed and resuspended in RPMI 1640 to the desired concentration.

Human blood was collected into 10 ml EDTA-anticoagulated tubes and mononuclear cells isolated by the Ficoll density gradient centrifugation method. The peripheral blood mononuclear cells (PBMC) were isolated from whole blood within 8 h of collection by centrifugation at 600 × g for 15 min through Ficoll-Hypaque Plus (Sigma–Aldrich). The white layer of cells at the plasma-Ficoll interface was harvested and washed three times with RPMI-1640 medium (Biosera, Nuaille, France) without fetal bovine serum (FBS). The cell count was measured with an automatic blood cell counter (Nihon Kohden, Tokyo, Japan). Cell viability was determined with a hemacytometer, using trypan blue exclusion method. Samples with the number of viable cells less than 95% and red blood cell contamination more than 2% were excluded from further tests.
This study was approved and monitored by National Research Ethics Committee, University of Tehran. All methods were performed in accordance with the relevant guidelines and regulations of the institution. Informed consent was obtained from subjects.

Peripheral blood was resuspended in RPMI 1640, layered with Ficoll Hypaque Plus (Sigma-Aldrich), and then centrifuged at 1000 g for 12 min. The red blood cell (RBC) pellet was incubated with red blood cell lysis buffer (CWBiotech, China) at a 9-fold volume for 15 min at 37°C to remove erythrocytes. After centrifugation at 1000 g for 10 min, the cell pellet was washed and resuspended in RPMI 1640 to the desired concentration. Neutrophil lysis was measured by lactate dehydrogenase (LDH) release assay. Briefly, the CFCM of S. aureus was added to 4.0 × 10⁶ neutrophils/mL to a total volume of 400 μL in 24-well plates and incubated at 37°C with 5% CO₂. At the desired times, the samples were centrifuged at 3000 rpm for 5 min, and the supernatants were collected. The LDH activity in the culture supernatants was measured by using an automatic biochemical analyzer 7600 (Hitachi, Japan) according to the manufacturer’s instructions.