Ecm cell adhesion array kit

ECM adhesion assay of control or NHE3kd cells (2 × 10⁵ cells/well, 2-h adhesion time) was performed using colorimetric ECM Cell Adhesion Array kit (Millipore, Cat. #ECM540) according to the manufacturer’s protocol.

These assays were performed as described previously (52 (link)). Briefly, the ECM Cell Adhesion Array Kit (ECM540, Millipore) was used to screen for MCL cell adhesion to the ECM according to the manufacturer’s manual. For specific cell adhesion assay, the plates were first coated with 10% FBS, fibronectin, or laminin, seeded with MCL cells pretreated with MI-2 at 0.5 μM for 30 minutes, and incubated for an additional 4 hours. The cells in suspension were thoroughly washed with PBS, and the resulting cells were lysed with Cell Titer-Glo Luminescent Cell Viability Assay Reagent (Promega). A BioTek Synergy HTX Multi-Mode microplate reader was used to quantify luminescence.

Cell adhesion to selected components of extracellular matrix (collagen I, collagen II, collagen IV, fibronectin, laminin, tenascin, vitronectin) was measured with the use of ECM Cell Adhesion Array Kit (Merck) according to the manufacturer protocol. Cells were detached from the wells and suspended in Assay Buffer. Then they were seeded on ECM protein-coated wells. After 2 h incubation, cells were rinsed with Assay Buffer, and Cell Stain Solution was added to each well. Wells were then washed, and Extraction Buffer was added to solubilise cell-bound stain. The absorbance was read at 570 nm on a microplate reader (iMark, Bio-Rad).

The assay was performed using the colorimetric ECM Cell Adhesion Array Kit (Merck Millipore, Germany) and also 96-well plates coated by ourselves. The ECM Array microtiter plates, pre-coated with different extracellular matrix proteins (Purified human Collagen I, Collagen II, Collagen IV, Fibronectin, Laminin, Tenascin, Vitronectin), were used according to the manufacturer’s instructions. Independently, 96-well culture microplates (Corning, USA) were coated with fibronectin (20 μg/ml) or laminin-1 (10 μg/ml), and incubated at 4 °C overnight. Non-specific binding sites on the plates were blocked by a one-hour incubation with 0.5% BSA in the RPMI medium at 37 °C in a CO₂ incubator. Next, the cells were counted and re-suspended in the culture medium to final concentration. Then, 50 μl of cell suspension of 4 × 10⁵ cells/ml were seeded in the wells (in triplicates) and allowed to adhere for 30–60 min at 37 °C. After that, unattached cells were removed by washing, and adherent cells were fixed with 4% PFA for 15 min and then stained with crystal violet solution (Sigma Aldrich, USA) for 10 min. After extensive washing, the dye was extracted using 2% SDS and quantified by spectrophotometry at 550 nm using a microplate reader (Multiskan RC, Labsystems, Thermo, Finland).

Cell adhesion assays were performed using the ECM Cell Adhesion Array kit (cat# ECM540, EMD Millipore) according the manufacturer’s instructions. 4175 cells were pretreated with 1 µM RAGE inhibitors (TTP488) or 0.1% DMSO for 48 h in culture media. Cells were detached from tissue culture plates using Accutase cell detachment solution (cat# AT104, Innovative Cell Technologies, Inc.), and 1.5 × 10⁵ cells/well seeded in triplicate to each well of the ECM Array Plate. Cells were then incubated for 2 h at 37 °C in a CO2 incubator. Cells were fixed in Cell Stain Solution (EMD Millipore) for 5 min, and the stain was extracted using Extraction Buffer (EMD Millipore), and total cell adhesion per well quantified in each by absorbance measurement at 595 nm (FLUOstar Omega microplate reader, BMG LABTECH). The measured absorbance was plotted as OD compared to both DMSO vehicle control, and a negative control well containing BSA.

Cells were cultured for 14 days with or without E2 (10 nM). The ECM Cell Adhesion Array kit (#ECM 540, Merck KGaA) was used according to the manufacturer’s instructions. Adhesion to collagen I and IV (#804592 and #C5533, Merck KGaA) was independently validated on collagen-coated (1 µg/cm²) 96-well plates. Cells (7 × 10⁴ cells per well) were allowed to adhere for 30 min. The wells were washed three times with PBS, fixed with cold methanol, and stained with 0.1% crystal violet. The adsorbed dye was extracted with a 10% acetic acid solution for 5 min, and measurement was performed at 595 nm on the Synergy2 microtiter plate reader (BioTek Instruments).