Ultra low cluster round bottom 96 well plates

Polymorphonuclear leukocyte (PMN) were isolated from 10–12 week old male murine bone marrow single cell-suspensions. Using density centrifugation and Histopaque® 1119 and Histopaque® 1077, the granulocyte layer was isolated from between the two density layers. After isolation, the neutrophils were rested on ice for 1 hr in D10 media (DMEM + 10% (vol/vol) FBS) before being transferred into ultra-low cluster round bottom 96-well plates (Costar) and incubated in an anaerobic chamber (0% O₂, 1.6% H₂, 37 °C) for 1 hour prior to experimentation. Cells isolated through this technique have been found to be 90–95% neutrophils (CD11b⁺Ly6G⁺) via flow cytometry.

Neutrophils were isolated as previously described (18 ). Bone marrow was flushed from the tibias and femurs of mice and single cell suspensions were created. Polymorphonuclear granulocytes were isolated using density centrifugation by isolating the neutrophils at the interface between the layers of Histopaque® 1119 and Histopaque® 1077. Neutrophils were resuspended in D10 media (DMEM, 10% FBS) and rested on ice for at least 1 hr before transferring to ultra-low cluster round bottom 96-well plates (Costar) in D10 media. Neutrophils were incubated (37 °C, 5% CO₂) for 1 hr prior to experiments. The isolated cells were 85–95% neutrophils (CD11b⁺Ly6G⁺).