Sample Collection. Blood was collected from 32 healthy volunteers (Supplementary file 1) in tubes containing EDTA-ACD (Acid Citrate Dextrose). The samples were centrifuged (250 g, 18 ○C, 15 min) to separate Platelet-Rich Plasma (PRP), the rest of the blood was diluted by normal saline (sterile, LPS-free). The PRP was spun (5000 g, 4 ○C, 20 min) and the upper AS was collected and refrigerated until the use in the cultures.
Neutrophil Isolation. After red blood cells (RBCs) sedimentation by dextran, the sample was decanted onto a 2-layered discontinuous density gradient of Percoll (86% and 55%) and centrifuged (480 g, 17 min, 18 °C, brake off). After centrifugation, the distinct mononuclear cells (on the Percoll 55%) and granulocytes (on the Percoll 86%) were removed separately. The neutrophils were washed and suspended in RPMI medium (Gibco).
For five of the samples, neutrophil isolation was performed by Percoll gradient (as above) as well as by Ficoll (Biosera) gradient centrifugation (25 min, 750 g, 18 ○C, brake off), followed by RBC lysis using hypo-osmotic shock.
The initial cell viability was evaluated by Trypan blue. The viability had to be ≥98% or the experiment would not be continued. In some cases, the viability obtained by Trypan blue was checked and confirmed by flow cytometry.
Cell Culture. To minimize the effect of variations in FCS/FBS products, we combined equal volumes of six product of FCS/FBS procured from different venders or lots and prepare a FCS/FBS mixture (one FCS product and two FBS products from Gibco plus two FCS products and one FBS product from Sigma). The mixture was used to supplement FCS cultures.
Neutrophils were cultured (Density: 5 × 105 cell/ml) in RPMI, which was supplemented by AP 10% or FCS 10% (the mentioned mixture), at 37 °C, CO25%, 90% humidity, for different times (12 h, 36 h and 60 h).
Cell Viability/Apoptosis Measurement. After the designated culture times, neutrophils were harvested, washed and resuspended in RPMI at 1×106 cell/ml concentration. Two aliquots of 200 µl were taken for further (CD11b and oxidative burst) analyses. The rest of the cells were stained using an Annexin-V-FITC Apoptosis detection kit (eBioscience) as per the manufacturer’s protocol and analyzed by flow cytometry.
CD11b Expression Assay. An aliquot of 2×105 neutrophils was stimulated with 100 ng/ml of endotoxin (LPS from Escherichia coli, serotype 0111: B4, Sigma) at 37 °C, CO2 5% for 30 min. Thereafter, the samples were stained with FITC anti-human CD11b mAb (Biolegend) or isotype control antibody (20 min at RT) and then run on flow cytometer.
Measurement of Oxidative Burst. 2×105 neutrophils were divided equally as experimental and negative samples, activated (or not for negative sample) by cell activation cocktail (Biolegend) for 20 min (37 ℃, CO2 5%), then dihydrorhodamine 123 (Santa Cruz) was added (final concentration of 1µM) and re-incubated for another 20 min. Then, the cells were placed into an ice bath (10 min), then washed and suspended in phosphate buffer saline containing formaldehyde 0.5% and analyzed by flow cytometry.
Flow cytometry was performed using a FACSCalibur flow cytometer (BD). Data were analyzed by FlowJo software version X.
Statistical Analysis. Statistical comparisons were estimated using repeated measurements analysis of variance (ANOVA), using IBM SPSS–25. The results are expressed as mean ± standard error of the mean (SEM). Differences were considered significant for P < 0.05.