I make use of a parallel plate flow chamber to study interaction of Mono Mac 6 cells (monocytic cell line) and HUVECs (human umbilical vein endothelial cells) under shear. My experiments include flow adhesion assays (continuous flow assays and detachment assays) at different flow rates, calculation of the rolling velocity and contact time as functions of the flow rate from captured video images, and analysis of the role of different receptors/ligands expressed on the endothelial membrane in rolling and firm adhesion of monocytes by using specific blocking antibodies. The flow chamber is connected to the flow circuit and positioned on an inverted phase contrast microscope (Zeiss Axiovert) stage. Flow is generated by a reciprocating pump.
HUVECs and MM6 cells are grown inside a 5% CO2 humidified incubator at 37 degrees C. Endothelial Growth Medium (EGM, Clonetics) and RPMI-1640 medium, supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic (Sigma), are used as the growth media for HUVEC and MM6, respectively. HUVECs (passages 3-5) are seeded in T-75 culture flasks or inside petri-dishes on sterilized gelatin- or fibronectin-coated glass slides.
MM6 cells are subcultured every 2-3 days in T-25 culture flasks to maintain a concentration of 0.5 million per ml. The number of cells is counted by a hemocytometer. Cell viability is determined by using trypan blue exclusion.
All steps relating to cell culture (apart from cell counting) are performed under sterile conditions.