Hetasep

To generate white blood cells (WBC), red blood cells were lysed from whole human blood, which was collected using either ethylenediaminetetraacetic acid (EDTA) or acid citrate dextrose (ACD) as a coagulant. Blood was mixed at a 1:10 ratio with ammonium-chloride‑potassium (ACK) lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA, pH 7.4) and incubated for 5 min at room temperature. The WBCs were pelleted by centrifugation (500 ×g, 5 min) at room temperature and then washed with cold phosphate-buffered saline (PBS). For the isolation of human neutrophils, whole blood was mixed with hetasep (Stemcell) in a 1:5 ratio and incubated for 30 min to separate leukocytes from erythrocytes. Subsequently, the straw-colored layer of leukocytes was transferred to a fresh tube and neutrophils were isolated using the direct human neutrophil isolation kit (Stemcell) according to manufacturer's instructions. Both WBCs and purified neutrophils were finally diluted in complete R10 media (RPMI-1640 media (Sigma) with 10% FBS, 2 mM L-Gluthamine and 100 U/ml penicillin/streptomycin) for each assay.

To isolate human neutrophils, peripheral blood was drawn into tubes containing trisodium citrate, citric acid, and dextrose (Vacutainer ACD Solution A, BD). Autologous serum, used for culture of cells was obtained by drawing blood into BD Vacutainer™ Venous Blood Collection Tubes SST, followed by centrifugation at 2500 × g for 30 min and serum collection. All blood donors provided written informed consent. For normal human blood samples, 10mls human blood was supplemented with GM-CSF (10 ng/ml) for 30 min at 37 °C followed by addition of 30 μg FITC-IgG isotype control or FcγRIIIB (3G8)-FITC-Ova conjugate for 2 h at the indicated concentrations. Blood was then incubated with Hetasep (STEMCELL Technologies) according to manufacturer protocols to deplete red blood cells and enrich leukocytes. Neutrophils were isolated from the leukocyte-rich plasma layer using a Easysep Neutrophil enrichment kit (STEMCELL Technologies). Neutrophil purity was evaluated using CD15, CD11b, CD66b, and lineage markers (CD3, CD19, and CD56) Supplementary Tables 4 and 6.

Peripheral blood was collected in two 10 ml sodium heparin tubes (14–18 ml of blood). Red blood cells were depleted with 1 volume HetaSep (Stem Cell Technologies) for 5 volumes whole blood and centrifuged at 92g, 6 min, no brake. The nucleated cells were layered on Histopaque 1077 (Sigma) and centrifuged (400g, 30 min, no brake). PBMCs were washed once with PBS before isolating cell types using magnetic microbeads. CD14+ monocytes were isolated with the Human CD14 Positive Selection Kit as previously described (EasySep™, Stem Cell Technologies) [50 (link), 51 (link)]. RNA was extracted from monocytes with the Qiagen AllPrep DNA/RNA Mini Kit (Qiagen, Chatsworth, CA). In parallel, neutrophils were isolated from the red blood cell/granulocyte pellet remaining after the Histopaque spin. The neutrophil pellet was washed once with PBS, and red cells were lysed with 5 ml water followed by 5 ml 2× PBS. Neutrophils were pelleted (300g, 10 min), lysed with STAT-60 (Tel-Test “B”, Friendswood, TX, USA), and RNA was extracted.

C2C12 cell myoblasts were purchased from ATCC (VA, USA) and were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) containing 10% fetal bovine serum (FBS, Gibco). At 80–90% confluence of cells, media was changed to DMEM containing 1% normal horse serum (NHS, Gibco) to differentiate C2C12 cell myoblasts to myotubes.
Isolation, culture and characterization of hUCB-MSCs were performed as described previously^{40 (link),41 (link)}. hUCB samples were obtained from the Seoul City Boramae medical center cord blood bank (Allcord) and obtained samples were harvested with the mother’s informed consent. The samples were mixed with Hetasep (Stem cell Technologies, Canada) at a ratio of 5:1 and incubated 1 hr at RT. Then, supernatant was collected with Ficoll and centrifuged 2,500 rpm, 20 min for separating mononuclear cells. The isolated cells were seeded at density 2 × 10⁵ cells per cm² on plates in growth media containing D-media (Formula 78–5470EF, Gibco, USA), EGM-2 singlequot and 10% fetal bovine serum (FBS, Gibco, USA). After 3 days, the non-adherent cells were washed out and the adherent cell colonies were cultured and grew to sharp, spindle shaped hUCB-MSCs. This process was performed under regulations and guidelines approved by the Institutional Review board (IRB) of Boramae medical center and Seoul national university (IRB.no 1608/001-021).

Peripheral blood was obtained from patients consented through the Moffitt Cancer Center Total Cancer Care protocol (MCC 14690/ Liberty IRB #12.11.0023) and approved by the Moffitt Cancer Center Scientific Review committee. Blood was treated with HetaSep™ (STEMCELL Technologies, Inc.) to remove the majority of red blood cells. Peripheral blood mononuclear cells (PBMCs) were isolated by ficoll separation. PBMCs (1 - 4 × 10⁵) were then plated in 1 mL of methylcellulose medium containing rhSCF, rhIL-3, and rhGM-CSF (MethoCult™ #H4534; STEMCELL Technologies, Inc.). All drug treated samples contained 0.1% DMSO as the final concentration. For healthy controls, 3 U/mL Epo was added. Cells were incubated at 37° C with 5% CO₂ and erythroid colonies were enumerated after 12–14 days. Data graphs and statistics were generated utilizing Prism (GraphPad Software).