Lymphocyte separation medium

This study was approved by the Institutional Review Board of the School of Medicine of Jinan University (JNUKY-2021-009 and JNUKY-2022-101). Peripheral blood samples (approximately 10 mL per sample) were extracted from NSCLC patients diagnosed at the First Affiliated Hospital of Jinan University (Table S1). The peripheral blood from the healthy donors were collected at Guangzhou Blood Center (200-400 mL per sample). Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using lymphocyte separation medium (GE, US) and stained with PE conjugated anti-human HLA-A2 antibody (BioLegend, Cat#343305, US) to identify the HLA-A2+ donors.

Approx. 20 mL peripheral blood were obtained from stroke patients and healthy controls in blood collection tubes containing ethylenediaminetetraacetic acid (EDTA) and PBMCs as well as neutrophils were isolated by density gradient centrifugation (lymphocyte separation medium; 1077 GE Healthcare). Serum was collected in reaction tubes, PBMCs in 50 mL centrifuge tubes. Neutrophils were obtained by lysing erythrocytes according to previously published methods [19 ]. Based on this method, activation of neutrophils is minor and prevented by the use of ion-free buffers. Serum samples were stored at −80 °C until further use, whereas PBMCs were used directly for flow cytometric (FACS) analysis and purified neutrophils for FACS and cell culture experiments.

Bone marrow mononuclear cells were isolated by density centrifugation using lymphocyte separation medium (GE Healthcare, Milwaukee, WI, USA). Cell cycle analyses were performed by incubating with 10 μg/ml Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C for 45 min, and 1.0 μg/ml Pyronin Y (Sigma, St. Louis, MO, USA) at 37°C for an additional 15 min. Cells were stained with mouse anti-human CD34-PerCP-Cy5.5 and CD38-APC-conjugated monoclonal antibodies (Becton Dickinson) at room temperature for 15 min.
To detect apoptosis, cells were incubated with CD34-PE, CD38-APC and CD45-V500 and then incubated for 15 min with Annexin-V-FITC and 7-amino-actinomycin D (7-AAD) apoptosis detection kit (Becton Dickinson) according to the manufacturer's instruction. Multi-parameter flow cytometric analyses were done using a BD LSRFortessa (Becton Dickinson). Aliquots of unstained samples were used as negative controls. Data were analyzed using BD LSRFortessa software (Becton Dickinson).

Primary human MФs from HD or patients were generated as described previously.^{27 (link)} BM mononuclear cells (BMMNCs) were isolated by density gradient centrifugation using a lymphocyte separation medium (GE Healthcare, Milwaukee, WI, USA). Briefly, monocytes isolated from BMMNCs of patients with PT, GGF or HD were differentiated into the cultivated PT MФs, GGF MФs or HD MФs for 7 days in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin, and 100 ng/mL macrophage colony-stimulating factor (PeproTech, Rocky Hill, NJ, USA). The resulting primary HD MФs were then polarized into M1 (BM-M1) or M2 (BM-M2) MФs by culturing for 24 hours in RPMI 1640 medium supplemented with either 100 ng/mL LPS and 20 ng/mL IFN-γ or 20 ng/mL IL-4 and 20 ng/mL IL-13, respectively. The cultivated BM MФs were evaluated with phagocytosis and migration assays as previously described.^{27 (link)} The supernatant of MФs and the BM plasma of patients with PT or GGF were harvested. The levels of TNF-α and TGF-β were measured using ELISA kit (Abcam, Cambridge, UK) following the manufacturer’s instructions.