Rpmi 1640 medium

The colorimetric assay used to assess the cell viability was based on the reduction of tetrazolium salt MTT (3-4,5-dimethylthiazole-2,5-diphenyltetrzolium bromide) by mitochondrial dehydrogenases to form an insoluble purple formazan. This test is suitable for measuring the number of viable cells, their activity, and proliferation [18 (link)]. We used H₂O₂-treated and -untreated granulocytes in the test. The granulocytes of patients and controls were isolated after separation of cell fractions by Lymphocyte Separation Medium (LSM, Capricorn Scientific GmbH, Ebsdorfergrund, Germany) and hypotonic lysis of precipitated erythrocytes. The cells were resuspended in an RPMI-1640 medium (Capricorn Scientific GmbH, Ebsdorfergrund, Germany) supplemented with 10% fetal calf serum (FCS) (SigmaAldrich, St. Louis, MO, USA), L-glutamine, and penicillin/streptomycin (Capricorn Scientific GmbH, Ebsdorfergrund, Germany) and incubated at 37 °C/5% CO₂ before MTT (5 mg/mL) was added. The cultures were incubated for 3–4 h at 37 °C. The reduction process was stopped by adding 10% SDS acidified with 1 N HCl. Dissolution of the resulting blue dye was continued overnight in an incubator. The absorbance was read at 540 nm on an ELISA reader (RT-6100, Rayto, Shenzhen, China). The ratio of the absorbances of treated to untreated living cells was used to measure the viability.

Two human glioblastoma cell lines, U-87 MG (Figure 1sa) and A-172 (Figure 1sb) cells were obtained from the national cell bank of Iran (Pasteur Institute, Tehran, Iran) and cultured in RPMI-1640 medium (Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin plus 100 μg/mL streptomycin (Gibco, Germany), under standard conditions (at 37 °C, 5% CO2 and 95% humidity). After culturing in 25 cm2 tissue flasks, the cells were trypsinized and then seeded into flat-bottomed adherent 48-well plates.

Ret cells [42 (link)] and BLM cells [43 (link),44 (link)] were cultivated in RPMI-1640 medium (Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 1%L-Glutamine, 10% FCS and 1% Penicillin/Streptomycin (Sigma-Aldrich St. Louis, MO, USA). Ret cell medium contained additionally 1% non-essential amino acid solution (Sigma-Aldrich St. Louis, MO, USA). HUVECs were freshly isolated as previously reported [45 (link)] and cultivated in EGM-2 medium (Lonza, Basel, Switzerland). Human embryonic kidney cells 293 (HEK 293) cells were cultivated in DMEM (Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 1%L-Glutamine, 10% FCS and 1% Penicillin/Streptomycin or for production of recombinant CHI3L1 in Opti-MEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1%L-Glutamine [46 (link)].

RPMI 1640 medium and DMEM (Dulbecco’s Modified Eagle Medium) were acquired from Capricorn Scientific GmbH (Ebsdorfergrund, Germany). L-glutamine, trypsin, and penicillin–streptomycin solution were from Biowest (Nuaillé, France). Thiazolyl blue tetrazolium bromide (MTT), fetal bovine serum (FBS), DMSO, Hoechst 33342, and carboxyfluorescein succinimidyl ester (CFSE) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). An Apoptosis Detection Kit based on Annexin-V-FITC (AV) and propidium iodide (PI) staining was acquired from Abcam (Cambridge, UK). Bovine serum albumin (BSA) was obtained from Serva (Heidelberg, Germany). Rhodamine 123 (Rho123) and tetramethylrhodamine ethyl ester (TMRE) were purchased from Sigma (St Louis, MO, USA). Carbonyl cyanide m-chlorophenyl hydrazine (CCCP, C2759) was purchased from Sigma-Aldrich (Darmstadt, Germany). FITC-conjugated anti-P-gp antibody was obtained from BD Biosciences (Winnersh, Berkshire, UK). Anti-P-gp mouse monoclonal antibody (Abcam, Cambridge, UK; ab10333) and secondary antibodies Alexa Fluor 555 goat anti-mouse secondary antibody (#A-21422, Thermo Fisher Scientific, Waltham, MA, USA) were obtained.

Heparinized peripheral blood was collected from 5 healthy volunteers and PBMCs were separated using a density gradient centrifugation protocol (Ficoll paque, sigma, USA) by a standard procedure. Briefly, blood samples were diluted 1:1 with PBS and transferred into a conical tube pre-filled with Ficoll paque solution. The cells were then centrifuged at 800 × g for 20 min at room temperature, and the PBMC layer was collected. Isolated cells were counted and resuspended in RPMI-1640 medium (Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin plus 100 μg/mL streptomycin (Gibco, Germany), and incubated at 37 °C in a humidified, 5% CO2 atmosphere. This study was approved by the Ethics Committee of Kerman University of medical sciences (98,000,781). All research was performed in accordance with relevant guidelines. Informed consent was obtained from all healthy donors.

At the age of 30 weeks, mice were weighed, and anesthetized by inhalation of isoflurane. The body size was measured by measuring the length from nose-tip to tail-base before euthanasia by neck dislocation. The procedures were in accordance with the animal experiment regulations of the Philipps-University Marburg (Ex 17/2022). Bone marrow-derived cells (BMDC) were isolated postmortem from femurs of mice as described by Amend et al. (35 ), cultured for 24 h in RPMI-1640 medium, containing 1% penicillin and streptomycin (Capricorn Scientific GmbH) and 10% heat-inactivated fetal bovine serum (FBS) (Capricorn Scientific GmbH) and in an environment of 37°C and 5% CO₂. For BMDC differentiation to bone marrow-derivate MΦ (BMDM), BMDC in the supernatant were centrifuged (250 ×  g; 10 min) and then cultured in RPMI-1640 (Capricorn Scientific GmbH) supplemented with 10% heat-inactivated FBS (Capricorn Scientific GmbH), 1% penicillin and streptomycin (Capricorn Scientific GmbH), and recombinant mouse GM-CSF (20 ng/ml) (BioLegend, San Diego, CA) and allowed to grow for 6 days. Incubation of BMDM of mice with IFN-γ and LPS for 24 h lead to the differentiation into BMDM1-MΦ, and incubation of BMDM with IL-4 and IL-13 for 24 h lead to the differentiation into BMDM2-MΦ.

The human leukemic monocyte cell line THP-1 (Leibniz Institute DSMZ, Braunschweig, Germany) was used. The THP-1 cells were frequently used as a model of monocyte/MΦ cell lineage (33 (link)) and routinely used in atherosclerosis research (29 (link), 34 (link)). THP-1 cells were cultured in RPMI-1640 medium (Capricorn Scientific GmbH, Ebsdorfergrund, Germany) supplemented with penicillin and streptomycin (Capricorn Scientific GmbH) as well as 10% fetal bovine serum (Capricorn Scientific GmbH). Cells were cultured at 37°C in a 5% CO₂ environment, with a medium change every 2–3 days. All experiments were performed using cells at the 9th passage or lower. Monocyte differentiation in M0-MΦ with 100 nM phorbol-12-myristate-13-acetate [PMA, (Sigma-Aldrich Chemie GmbH Munich, Germany)] was performed as described by Ackermann et al. (34 (link)).