Human chemokine array

WT or KO BEAS-2B cells were seeded in 12-well plate (1.3 × 10⁵/well) and cultured in Dulbecco's modified Eagle's medium (DMEM) with 5% FBS. After 24h, media were aspirated, and cells were washed with PBS, followed by addition of 1 ml of serum-free DMEM. After 24h, cell culture media were collected and then centrifuged at 1,500 rpm for 10 min at 4°C. The levels of chemokines were determined by Human Chemokine Array (R&D systems, Minneapolis, MN) according to manufacturer's protocol.

Naïve neutrophils were isolated using the MACSexpress (Miltenyi Biotec, Bergisch Gladbach, Germany) whole blood isolation kits. Purity of neutrophils was confirmed by Wright Giemsa staining (Thermo Fisher, Waltham, MA, USA) and flow cytometry. The purity was close to 99%. Overall, 10⁹ neutrophils were treated with CM61 EVs or HM59 EVs for 2′, 10′ 30′ and 60′ in HBSS buffer. Supernatants and neutrophils were collected post treatment along with untreated controls and controls that were incubated in HBSS for 60′. Then, 1000 ug of cell supernatants from the 2′, 10′, 30′, and 60′ neutrophils treated with CM61 EVs were subjected to the chemokine analysis using the Human Chemokine array (R&D Systems, Minneapolis, MI, USA). Arrays were quantified using ImageJ software (NIH, DC, USA).