Cytoselect cell migration assay kit

To differentiate HL-60, cells were cultured in medium containing 1.3% DMSO for 5 days, as previously reported [20 (link)]. Differentiated HL-60 (dHL-60) cells were washed twice with serum free medium and chemotaxis assays were performed using 3 µm CytoSelect™ Cell Migration Assay Kit (Cell Biolabs, San Diego, CA, USA), according to the manufacturer’s instructions. Briefly, 2 × 10⁵ cells/well were seeded onto the upper membrane chamber and cell culture supernatants from HaCaT cells incubated with M5 and various concentrations of PG102 were added to the bottom wells. After 2 h, migrated cells in the bottom wells were lysed and fluorescence was read at 480 nm/520 nm using FlexStation 3 microplate reader (Molecular Devices, San Jose, CA, USA).

Primary arterial and venous VSMCs from control and RP105^−/− mice were grown to confluence and then made quiescent in cultured medium supplemented with 1% FCS for 24 hours. Cells were detached from the surface using Accutase Cell Detachement Solution (Innovative Cell Technologies, Inc., San Diego, CA, USA) and suspended at a concentration of 100.000 cells/ml in culture medium supplemented with 1% FCS. Migration was assayed with a polycarbonate membrane inserts having 8 μm-pores in 24-well chemotaxis chambers using commercial CytoSelect Cell Migration Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA) over 16 hours towards the 20% FCS gradient. All migratory cells were lysed and labeled with fluorescent dye (CyQuant GR). Quantification was performed on a fluorescence plate reader at 480 nm/520 nm.

Cell migration a was determined using the CytoSelect Cell Migration Assay kit (Cell Biolabs, San Diego, CA, USA), according to the manufacture’s protocol. Briefly, T cells (CD4+ cells, 1 × 10⁶) from the spleen and macrophages (F4/80+ cells, 1 × 10⁶) from the liver were isolated, and cells were loaded into the upper chamber of the two systems. The lower chambers were loaded with 300 ng/ml recombinant mouse CCL2 (BioLegend) or vehicle (PBS). After 6 h of stimulation, cells that had migrated were stained using CyQuant GR dye solution, and fluorescence intensity was measured at 480 nm/520 nm. Data are shown in relative fluorescence units.

Human neutrophils were isolated from peripheral blood of healthy adult donors using Cytoselect Cell Migration Assay kit (Cellbiolabs Inc., San Diego, CA). Neutrophils were suspended in HBSS at the concentration of approximately 1.5×10⁶ cells/ml and 300 µl were loaded into the top chamber of a 24-well Boyden chamber plate (Cell Biolabs inc., San Diego, CA). Sterile PBS or a bacterial suspension (1.5×10⁷ CFU/ml) in chemotaxis medium was added to the bottom chamber. In some experiments, the complement inhibitor Futhan (6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate) [34] (link) was added to the bottom chamber at a final concentration of 50 µg/ml. Human IL-8 (100 ng/ml, R&D Systems) or fMLP 10 nM (N-Formyl-L-methionyl-L-leucyl-L-phenylalanine, 10 nM) (Sigma, St. Louis, MO) were also added to the bottom chamber in some experiments. In some experiments, PBS (vehicle control) or mouse monoclonal anti-human C5a antibody (ab11876, Abcam) dissolved in PBS was added to the bottom chamber at a final concentration of 10 µg/ml. The plates were incubated for two hours at 37°C in tissue culture incubator. The cells that migrated to the bottom chamber were counted using a hemocytometer. Each experiments was repeated with cells from at least 3 different donors.

Cytoselect cell migration assay kit was purchased from Cell Biolabs, Inc and the assay was performed according to the manufacturer's instructions. Briefly, DU145 cells (75×10³) were seeded in transwells (8 μM pore size inserts) in serum-free media and were stimulated with recombinant HGF or with conditioned media from 18Co or WI38 fibroblasts (in the absence or the presence of SRI 31215 or JNJ 38877605). Cells were incubated for 12 hours and non-migratory cells were gently removed from inside of the inserts. Inserts were stained for 10 minutes at room temperature, transferred to wells containing 200 μl of extraction buffer and incubated for 10 minutes on a shaker. The optical density (OD) was measured at 560 nm.

A scratch in the cell monolayer was performed and migration extent was evaluated at 48 hours after healing. Transwell migration assay was performed using the CytoSelect^™ Cell Migration Assay Kit, according to manufacturer's indications (Cell Biolabs, Inc).