A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. relevant shear flow1 2 In this paper we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions the microfluidic channel surface was coated with endothelial cells (ECs) which were then activated with tumor necrosis factor-α (TNF-α) significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform that permits rapid analysis of parameters such as rolling velocities and rolling path are important advantages for assessing cell rolling properties P-and E-selectin (P-and E-sel) and their counter ligands on the surface of leukocytes5 6 Better understanding and improved efficiency of cell homing and specifically the rolling step are of great importance in the quest for new platforms to improve cell-based therapy. To date this has been achieved by using parallel plate flow chambers (PPFCs) comprising two flat plates with a gasket between them with an inflow and outflow port located on the upper plate through which a cell suspension is perfused by using a syringe pump7 8 9 The surface of the bottom plate can be coated with a relevant cell monolayer/substrates and the conversation between perfused cells and the surface under shear flow is then explored7. However PPFC is a low throughput reagent-consuming and fairly tedious method with bubble formation leakage and poorly controlled flow presenting major drawbacks. An alternative technique to the traditional PPFC is usually a multi-well plate microfluidic system permitting higher throughput performance of cellular assays (up to 10 times higher than PPFCs) under accurate computer-controlled shear ADX-47273 flow with low reagent consumption1 10 Cell rolling experiments are performed inside the microfluidic channels which can be coated with cell monolayers or engineered substrates and imaged using a microscope with rolling properties readily analyzed using a suitable software. In this study we demonstrate the capabilities of this multi-well plate microfluidic system by studying the rolling properties of human promyelocytic leukemia (HL-60) cells on different surfaces. HL-60 rolling on substrates like P-and E-sel as well as on cell monolayers expressing different rolling receptors was analyzed. In addition antibody (Ab) blocking was used to demonstrate direct involvement of ADX-47273 specific selectins in mediating the rolling movement of HL-60 on those surfaces. Rolling experiments were performed with increased throughput under stable shear flow with minimal reagent/cell consumption allowing efficient analysis of key rolling parameters such as rolling velocity number of rolling cells and rolling path properties. Protocol 1 Cell Culture Human promyelocytic leukemia (HL-60) cells Culture HL-60 cells in 75 cm2 flasks with 15 ml of Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 20% (v/v) fetal bovine serum (FBS) 1 (v/v) L-Glutamine and 1% (v/v) Penicillin-Streptomycin. Change media every 3 days by aspirating half of the cell suspension volume and replacing it with complete IMDM media. For carboxyfluorescein diacetate succinimidyl ester ADX-47273 (CFSE) staining centrifuge HL-60 cell suspension (400 x g 5 min) resuspend in a 1 μM CFSE solution (prepared in prewarmed PBS) and incubate for 15 min at 37 °C. Then centrifuge cells aspirate supernatant and resuspend cells in fresh prewarmed medium for 30 min. Wash cells in PBS and then Rabbit Polyclonal to ARHGEF11. use for rolling experiments (see Physique 1B for representative image of CFSE-stained HL-60 cells on P-sel-coated surface). Note: CFSE staining is usually optional ADX-47273 and is presented here to demonstrate the rolling phenomenon in the microfluidic channel. Analysis of rolling parameters presented in this manuscript was performed on unstained cells using standard brightfield imaging. Lung microvascular endothelial cells (LMVECs) Coat 100 mm Petri dishes with 0.1% gelatin solution (v/v in PBS) and incubate at 37 °C for at least 30 min. Culture LMVECs on gelatin-coated 100 mm Petri dishes in complete endothelial growth medium (endothelial basal medium-2 (EBM-2)) supplemented with a specific growth supplement kit see REAGENTS). Change media every other day and sub-culture cells upon reaching 80-90% confluence. For sub-culture wash cells with PBS and then.