Macsxpress whole blood human neutrophil isolation kit

Fresh blood in EDTA vacutainer tubes from healthy donors was kindly provided by the Blood Collection Center for Research, USAISR Donor Center. Neutrophils were isolated within 1 h of blood draw using the MACSxpress whole blood (human) neutrophil isolation kit (Miltenyi Biotec, Auburn, CA) and red blood cell lysis was performed using erythrocyte depletion kit (Miltenyi Biotec) per manufacturer’s instructions. Isolated neutrophils were washed once with Hank’s balanced salt solution without Ca²⁺ and Mg²⁺ [HBSS] (Gibco-Life Technologies, Carlsbad, CA, USA) and suspended in media containing RPMI1640 (ATCC, Manassas, VA, USA) supplemented with 0.5% human serum albumin (Celprogen Inc., Torrance, CA, USA)⁶⁶ and 1% penicillin–streptomycin (Gibco-Life Technologies). Cell viability was determined by trypan blue exclusion method using a TC20 automated cell counter (Bio-Rad Laboratories Inc., Hercules, CA, USA). After staining the cells with the CD15 & CD16 antibodies (Miltenyi Biotec) per manufacturer’s instructions, neutrophil purity (Supplementary Figure S1; mean ± SD is 96.6 ± 1.6) was tested using the Attune NxT flow cytometer (Invitrogen-Life Technologies, Carlsbad, CA USA), Majority of the experiments/assays were performed with blood from different donors on different days.

Whole blood was collected from healthy donors by the “Ecole française du sang” (EFS, Toulouse Purpan, France) in accordance with relevant guidelines. Written, informed consent was obtained from each donor. Neutrophils were then isolated by negative selection using MACSxpress Whole Blood Human Neutrophil Isolation Kit (Miltenyi Biotech) according to manufacturer’s instructions. Following isolations cells were centrifuged 10 min at 300 g and red blood cells were eliminated using Red blood cells (RBC) Lysis Buffer (BioLegend). This procedure gives >95% of neutrophil population as assessed by flow cytometry of CD15+/CD16+ cells.