2.5. Isolation of Mouse Primary Peritoneal Macrophages and Splenocytes

Mouse primary peritoneal macrophages were isolated using the method as described [30]. Briefly, peritoneal macrophages were isolated by lavaging the peritoneal cavity of experimental mice with 2 aliquots of 5 mL sterile Hank’s balanced salts solution (HBSS) (50 mL of 10× HBSS (Hyclone, SH30015.02, South Logan, UT, USA), 2.5 mL of penicillin–streptomycin–amphotericin solution (PSA, 100×, Biological Industries, 03-033-1B, Kibbutz Beit Haemek, Israel), 20 mL of 3% bovine serum albumin (BSA, Sigma-Aldrich Co., A9418, St. Louis, MO, USA) in phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.4, 0.22 μm filtered), 2.0 mL of 7.5% NaHCO₃ (Wako, 191-01305, Osaka, Japan) and 425.5 mL sterile water) for a total of 10 mL through peritoneum. The peritoneal lavage fluid was collected and centrifuged at 400× g for 10 min. The cell pellet was isolated and resuspended in tissue culture medium (TCM, a serum substitute, Celox Laboratories, Lake Zurich, IL, USA). TCM medium consisted of 10 mL TCM, 500 mL Roswell Park Memorial Institute (RPMI) 1640 medium (Atlanta Biologicals Inc., Norcross, GA, USA) and 2.5 mL of antibiotic–antimycotic solution (100× PSA). Isolated peritoneal cells are macrophages that can serve as a cell culture model for assessing inflammation status in vitro. The viable cell number was counted under a microscope with a hemocytometer using the trypan blue exclusion method. The macrophages cell density was adjusted to 2 × 10⁶ cells/mL TCM medium for use.

After peritoneal macrophages were collected, the mouse spleen was cut aseptically, immersed in TCM medium and ground to isolate splenocytes [31]. Splenocytes were then collected and centrifuged at 400× g for 7 min. Next, the splenocytes were resuspended in an aliquot of 10 mL of red blood cell (RBC) lysis buffer (pH 7.4, 0.22 μm filtered) consisting of 0.017 M Trizma Base (Sigma-Aldrich Co., St. Louis, MO, USA) and 0.144 M NH₄Cl (Sigma-Aldrich Co., St. Louis, MO, USA) in sterile water. After standing for 3 min, the cell solution was centrifuged at 400× g for 7 min. The splenocyte pellet was carefully washed with HBSS three times. Isolated splenocytes were resuspended in a 3 mL TCM medium. The splenocytes were computed using the trypan blue dye exclusion method using a hemocytometer. Finally, the splenocytes cell density was adjusted to 1 × 10⁷ cells/mL TCM medium for use. Isolated splenocytes are approximately composed of 41.54% of B lymphocytes and 47.11% of T lymphocytes, as well as trace antigen-presenting cells that are suitable cell cultures for evaluating Th1/Th2 immune responses in vitro [32].