Biotek microtiter plate reader

Chemotaxis assays were performed using the ChemoTx System from NeuroProbe with modification (NeuroProbe, Gaithersburg, MD). Briefly, samples of rhBD-2 were prepared at 3 concentrations (100 ng/μl, 200 ng/μl and 400 ng/μl) selected following kinetic analysis of optimum chemotaxis for CEM-SS cells, then incubated for 72 hr at 37°C in either 10μM MGO or 0.0067M PBS, pH 7.5. CEM-SS cells were initially grown in RPMI media, then resuspended in “chemotaxis media” (serum-free HG-DMEM with 1% BSA). The chemotactic mix containing rhBD-2 at final concentrations of 0, 10, 20 and 40 μg/ml, with or without MGO, or 10 nM stromal cell-derived factor 1 (SDF-1) as positive control (shown previously to induce a chemotactic response in CEM-SS cells, unpublished) were added to lower wells (30 μl) of a ChemoTx 96-well chamber. CEM-SS cell suspension was loaded into top wells of the chamber for a final cell concentration of 1 x 10⁶ cells/ml, and the chamber incubated for 2 hr in 5% CO₂ at 37°C. After incubation CyQuant cell dye (Life Technologies, Grand Island, NY) was injected into the lower sample wells, and developing fluorescence quantitated using a BioTek microtiter plate reader (BioTek, Winooski, VT).

We studied the activation of plasminogen (human, murine, or a mixture of both) by SAK in the absence or presence of α₂AP, fibrin and S. aureus bacteria. Human and murine plasminogen were isolated from plasma by lysine Sepharose affinity chromatography, as described previously [44 (link)]. SAK variant TS-162 was previously described [45 (link)]. Murine α₂AP was obtained from Abcam (Cambridge, UK). SAK, α₂AP and plasminogen were diluted in 0.1M sodium phosphate buffer, pH 7.4, containing 0.05 M NaCl and 0.01% Tween. Solid fibrin clots were formed upon addition of bovine thrombin (1 U/mL) to human fibrinogen (200 μg/mL in 0.05 M Tris-HC1 buffer, pH 7.4, containing 0.038 M NaCl and 0.01% Tween 80) (Calbiochem, EMD Millipore, Billerica, USA) (30 min, 37°C). CNBr-digested murine fibrinogen (Fg(CNBr)) was prepared as published [46 (link)]. In some experiments, S. aureus bacteria (OD₆₀₀ 2.0, 15% vol/vol; live or heat-killed at 60°C for 1h) were used in the reaction mixture. In this case, bacteria were pelleted by centrifugation before read-out of the absorbance at 405 nm (A₄₀₅). Hydrolysis of the chromogenic substrate S-2403 was used to monitor plasmin activity in a Bio-TEK microtiter plate reader (Bio-TEK, Winooski, USA).

MDA-MB-231 cell invasion was determined using the CytoSelect Cell Invasion Assay (Cell Biolabs, Inc., San Diego, CA) according to manufacturer’s instructions. Briefly, MDA-MB-231 cells were pre-treated with (-)-oleocanthal for 24 h. Basement membranes of Boyden chambers were rehydrated with 300 µl serum free RPMI-1640, and 3×10⁵ cells were then seeded into the upper area of the chamber in serum free RPMI-1640. Bottom wells were filled with defined control serum-free media supplemented with 40 ng/ml HGF containing (-)-oleocanthal or no (-)-oleocanthal. After 24 h incubation (37°C, 5% CO₂), non-invasive cells were removed from the upper chamber and cell invasion was assessed by light microscopy after staining of invaded cells with crystal violet Cell Stain Solution (Cell Biolabs, CA). For colorimetric quantification of invasion, inserts were then placed in extraction buffer (200 µl, 10 min), and absorbance at 560 nm was determined after transfer to a 96 well plate (100 µl per well) using a BioTek microtiter plate reader (BioTek, VT).