Red blood lysis buffer

Leukocytes were isolated from whole peripheral blood via centrifugation (1,500 g for 15 min at 4°C) after lysing the red blood cells with Red Blood Lysis Buffer (Solarbio, China). RNA from the three paired samples was extracted with RNeasy Mini Kit (Qiagen, Germany) per the manufacturer's instructions. RNA from the other 44 samples was extracted using Sparkzol Reagent (SparkJade Science Co., Ltd., China), chloroform, and isopropanol precipitation. The RNA Integrity Number (RIN) of the three paired samples was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA); all samples had RIN ≥7.5. The integrity of all 50 RNA samples was determined by agarose gel electrophoresis; RNA concentration and purity were quantified with a Nanodrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA). The RNA samples were stored at −80°C until further use.

Immediately after euthanasia, 1 ml aliquots of PBS were slowly infused in the murine lungs through the tracheostomy and then withdrawn gently. This lavage was repeated three times using the same syringe. The collected lavage fluid was stored in a 10 ml tube on ice. The fluid was centrifuged at 1000 rpm and 4 °C for 10 min, and the cell sediment was washed with PBS. The cell-free supernatant was centrifuged again at 14,000 g and 4 °C for 10 min, stored at − 80 °C and used for determination of cytokines content via ELISA. To the pellet, red blood lysis buffer (Solarbio, China, R1010) were used for 15 min and washed with PBS. Next, the pellet was resuspended for analysis.

Plasma samples were procured from healthy volunteers with written informed consent. Human peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by Ficoll-Hypaque (Tianjin Haoyang, China) density centrifugation, washed with PBS, suspended in red blood lysis buffer (Solarbio, China) at 4 °C for 15 min. Wash again and then suspended in PBS for tail vein injection into NPG mice.