Eosinophil isolation kit

Blood was drawn from healthy, nonatopic volunteers into syringes containing 10% 0.1 M citrate and was immediately processed. Cells were separated by Ficoll gradient centrifugation (Lymphoflot no. 824012; Bio-Rad Laboratories) according to the manufacturer’s specifications. From now on, all procedures described were performed at 4°C. The granulocyte-rich red pellet was subsequently lysed using ice-cold distilled water. Untouched eosinophils were isolated using a commercially available eosinophil isolation kit (130-092-010; Miltenyi Biotec) according to the manufacturer’s specifications. Purity of enriched eosinophils was evaluated by flow cytometry of side scatter and CD16 expression. Cells were resuspended in RPMI medium containing 10% FCS and stimulated with ADP or baicalein for 30 min at 37°C. Then, cells were washed and resuspended in HBSA buffer, and cell lysates were generated by three repeated freeze-thaw cycles. Samples were stored at −80°C until further analysis.

Eosinophils were isolated from the peripheral blood of healthy and asthmatic individuals. Venous blood (120 mL) was collected with EDTA and mixed with HBSS in a 1:1 ratio. Separation of the granulocytes and erythrocytes from the mononuclear cells was done by density gradient centrifugation with Histopaque 1077 (Sigma–Aldrich, St. Louis, MO). Dextran sedimention and hypotonic lysis were used to remove red blood cells. Eosinophils were purified using negative selection in a cocktail of antibodies provided in Eosinophil Isolation Kit (Miltenyi Biotec, Auburn, CA), with autoMACS (Miltenyi Biotec, Auburn, CA). Purity (>99%) and viability (>98%) of eosinophils were determined by staining with Hema-Diff (StatLab, Lewisville, TX), and Trypan Blue (Sigma–Aldrich), respectively.

Blood from SA and non-severe asthma (non-SA) patients were layered onto a Lymphoprep™ solution (Axis‐Shield, Oslo, Norway), followed by centrifugation at 2500 rpm at 20 °C for 25 min without braking. The layer containing peripheral blood mononuclear cells was collected; after red blood cells were completely eliminated by hypotonic lysis, monocytes were isolated for further experiments.
The layer containing red blood cells and granulocytes was sedimented in Hank’s balanced salt solution (HBSS) containing 2 mM ethylenediaminetetraacetic acid (EDTA) and 2% dextran at 37 °C for 45 min. The neutrophil‐rich layer was collected, and red blood cells were lysed. Then, PBEs were isolated from the neutrophil‐rich layer according to the manufacturer’s protocol. PBNs were identified as magnetically labeled non-eosinophils and collected by eluting the retained cells. Isolated PBNs were maintained in RPMI‐1640 medium supplemented with 2% heat‐inactivated fetal bovine serum. Monocyte Isolation Kit II, Eosinophil Isolation Kit, and MACS Columns were purchased from Miltenyi Biotec Inc. (Auburn, CA, USA). Cell purity (>95%) was assessed by H&E staining and flow cytometry based on CD68 and CD11b expression in neutrophils, Siglec‐8, and eosinophil cationic protein (ECP) expression in eosinophils, and CD14 expression in monocytes^{23 (link)}.

Eosinophils were isolated from healthy controls using the Eosinophil Isolation Kit (Miltenyi Biotec Inc). Briefly, blood was layered on the Lymphoprep (Axis‐Shield), followed by density gradient centrifugation at 2000 rpm at 20°C for 25 minutes without brakes. The granulocyte fraction was lysed for red blood cells by using cold distilled water for 30 seconds. We collected unlabeled cells that pass through. Cell purity (>5%) was evaluated by flow cytometry based on Siglec‐8 and ECP expression for eosinophils, and cell viability evaluated after vitamin E treatment was more than 90% at 800 μmol/L (data not shown).

Peripheral blood neutrophils (PBNs) from 4 asthmatics were collected into BD Vacutainer ® tubes containing acid citrate dextrose solution (BD Biosciences, Franklin Lakes, NJ, USA). As previously described,¹⁹ (link) blood was layered onto Lymphoprep™ solution (Axis Shield, Oslo, Norway) and centrifuged at 2000 rpm at 20 °C for 25 min without braking. The layer containing granulocytes and red blood cells (RBCs) was sedimented for 30 min in 2% dextran, diluted in Hank's balance salt solution (HBSS) buffer supplemented with 2 mM ethylenediaminetetraacetic acid (EDTA) at room temperature. The upper layer was harvested and washed once with HBSS buffer supplemented with 2 mM EDTA. The eosinophils contaminated were excluded by using the Eosinophil Isolation Kit and MACS Column (Miltenyi Biotec Inc, Auburn, CA, USA) according to the manufacturer's protocols. Cell viability (>98%) and purity (>95%) were assessed by Trypan blue staining and flow cytometry based on CD11b (neutrophil marker). The isolated PBNs were suspended in the culture medium for 30 min prior to further experiments.

Whole‐blood components were separated by density gradient centrifugation. The erythrocyte pellet was lysed, and eosinophils were isolated with the Eosinophil Isolation Kit in an unlabeled manner (Miltenyi, Bergisch Gladbach, Germany).