Interleukin 4 (il 4)

The non-polarized or M0 phenotype was achieved by cultivating cells in RPMI-1640 medium supplemented with 10% FBS (Cat. No. ES-009-B; Millipore, Burlington, MA, USA), 10% MCSF (L929-CM), 1% PEST, and 1% L-glutamine for 72 h. The media was supplemented either with 50 ng/mL recombinant mouse IFNγ (Cat. No. BMS326; eBioscience, San Diego, CA, USA) and 100 ng/mL Pam3SCK4 (Cat. No. tlrl-pms; InvivoGen, San Diego, CA, USA) to achieve M1 phenotype, or with 20 ng/mL IL-4 (Cat. No. RP-8666; Invitrogen, Waltham, MA, USA) to achieve M2 phenotype.

The femurs and tibias were isolated from euthanized C57BL/6 mice (4–6 weeks of age) aseptically. After removing muscles, the bones were flushed with Dulbecco’s Modified Eagle’s medium (DMEM, Gibco) using syringe (26G × ½ needle) to extrude bone marrow at least 3 times. After centrifugation of bone marrow, pellet was re-suspended with 1.0 ml of ammonium-chloride-potassium (ACK) lysis buffer (Gibco) to lyse the red blood cells, and supernatant was aspirated from the white cell pellet after centrifugation. Cells were cultured with DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1% antibiotic-antimycotic (Gibco) (10% FBS DMEM) and 10 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) for 5 days to obtain Bone Marrow-Derived Macrophages (BMDMs). Additionally, cells were cultured for 6 days by adding 100 ng/ml IL-4 (Invivogen) to the above media to prepare Bone Marrow-Derived Dendritic Cells (BMDCs). Whole peripheral blood obtained from mice was diluted with roswell park memorial institute (RPMI) 1640 medium (Gibco) and PBMCs were isolated by Histopaue-1077 (Sigma). Isolated PBMCs were washed 3 times and cultured in 10% FBS and 1% antibiotic-antimycotic included RPMI1640 medium (10% FBS RPMI).

BALB/c or C57Bl/6 mice were euthanized by rapid cervical dislocation, femurs were gently isolated and washed with PBS. Bone marrow cells were flushed out of the bones, washed twice with PBS. Erythrocytes were lysed using ACK Lysis Buffer (Gibco part of Thermo Fisher, Waltham, MA, USA), similarly as in the case of splenocytes. Next, cells were counted and plated on a sterile Petri dish (2 × 10⁵ cells/mL) in 10 mL of macrophage complete medium DMEM/F-12 (Gibco), supplemented with 10% FBS (HyClone), 1% pen-strep (Sigma Aldrich) and 20% L929 CM. The fresh full macrophage medium was changed every 3 days. When indicated, BMDMs were stimulated with lipopolysaccharide (LPS) or IL-4 (Invivogen, San Diego, CA, USA) for 4, 24, 48, or 72 h. Next, cells were detached and stained for flow cytometry.