Easysep mouse monocyte enrichment kit

Leukocyte pools from C57BL/6 WT or Ltb4r1 KO mice, bled by retro orbital sinus, were subjected to RBC lysis buffer and monocyte subsets were enriched with EasySep® mouse monocyte enrichment kit (STEMCELL tech, Vancouver, BC) following the manufacture’s instructions. Isolated monocytes (2×10⁶ to 5×10⁶) were washed once in serum-free medium (RPMI-1640) and suspended in 2 ml of Diluent solution C (included in the PKH26 labeling kit). Two ml of PKH26 (Sigma Chemical Co. St Louis, MO) at 2×10⁻³ M in Diluent C was added and mixed, and the cells were incubated for 10 min at room temperature in the dark. The staining reaction was stopped by addition of an equal volume (2 ml) of medium supplemented with 10% FBS. The mixture was centrifuged and the cells were washed once and resuspended in serum containing medium. Subsequent to labeling with PKH26, the monocytes were counted and ~0.5×10⁶ viable cells were suspended in 0.2 ml PBS and i.v. injected into retroorbitally in each group of mice. Five days after injection, SVCs isolated from visceral fat tissue and analyzed by FACS.

To obtain a pure population of splenic monocytes, intact spleens were rapidly excised from anesthetized WT or Alox15-deficient mice and ruptured between two microscope slides. The spleen was then rubbed through a 40 micron cell strainer and rinsed with Pharmlyse to lyse red blood cells. After 5 min incubation in the dark at room temperature, the strained cells were washed with PBS containing 2 mmol/L EDTA and 0.5% BSA (MACS buffer). An EasySep mouse Monocyte Enrichment Kit (Stemcell Technologies) was then used to deplete non-monocyte populations by antibody-biotin-magnetic bead separation. The remaining population was confirmed by flow cytometry to be approximately 93% pure Ly-6C⁺CCR2⁺ monocytes. After isolation of splenic monocytes, cells were washed with Hank’s Balanced Salt Solution to remove EDTA and to supplement calcium. Monocytes were then divided (1 × 10⁵ cells/incubation) and stimulated with calcium ionophore (A23187; 10 µmol/L) and docosahexaenoic acid (DHA; 10 µmol/L) for 30 min at 37°C. The incubation was then terminated with 1.5 mL of ice-cold methanol and the samples were immediately frozen at −80°C for subsequent LC-MS/MS analysis (see above).

After red blood cell lysis, circulating monocytes from C57/BL6 WT, Ltb4r^–/–, ACTb^EGFP, or Ccr2^RFP/RFP mice were enriched with EasySep mouse monocyte enrichment kit (STEMCELL Technologies) following the manufacturer’s instructions.