Mba 96

Chemotaxis of BMDM was performed using a modified Boyden chemotaxis chamber containing a 96-well microchemotaxis plate (MBA-96; Neuro Probe, Cabin John, MD) as described previously with minor modifications [70 (link)]. Briefly, the bottom wells of the chamber contained 40 μl of HA6 (4 mM) dissolved in defined medium (DM), without FCS. Positive and negative controls included 10% FCS or DM, respectively. A 5-μm-pore polycarbonate membrane filter was placed between the bottom and top chamber. The upper wells were filled with 1 × 10⁶ cells/ml suspended in 10 μl DM. The chamber was incubated for 6 h at 37 °C. Non-migratory cells on the upper surface of the membrane were treated with 200 μl of 1 mM EDTA for 15–20 min and wiped off. Cells that had migrated into the membrane were stained with Diff-Quick (Baxter Healthcare, McGaw Park, IL) and counted in five randomly selected high-power fields in each well. Each chemoattractant solution was tested in six wells and each experiment was repeated at least three times. Data were expressed as the number of macrophages that migrated into the membrane for each condition and converted to a percentage of control (DM) and data from three separate experiments were combined.