Adhesion assays were performed using the Vybrant Cell Adhesion Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, CAR-T cells were washed twice with phosphate-buffered saline (PBS) and then resuspended in RPMI at 5×106 cells/mL. Calcein AM stock solution was added at a final concentration of 5 µM. Samples were mixed well and incubated at 37°C for 30 min. After labeling with calcein AM, CAR-T cells were washed twice and resuspended in plain RPMI at 1×106 cells/mL. Subsequently, 100 µL of the calcein-labeled CAR-T cell suspension (1×105 cells unless otherwise indicated) was added to the prepared microplate wells containing confluent endothelial cell monolayers and incubated at 37°C for 90 min. The plates were washed four times to remove non-adherent calcein-labeled cells and then 200 µL of PBS was added to each well and fluorescence was measured. Prior to T-cell addition, target cells (where indicated) were exposed to bovine serum albumin (BSA) or VEGF-A or VEGF-A/anti-VEGF-A antibody complexes.
Vybrant cell adhesion assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The Vybrant Cell Adhesion Assay Kit is a laboratory equipment product designed to measure cell adhesion. It provides a quantitative assessment of cell-to-cell or cell-to-extracellular matrix interactions.
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30 protocols using vybrant cell adhesion assay kit
1
Adhesion Assay for CAR-T Cell Binding
2
Quantifying Cell Adhesion Dynamics
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Adhesion assays were performed by using the Vybrant™ Cell Adhesion Assay Kit (V-13181, Thermo Fisher Scientific) according to the instructions. Briefly, cells (3.5 × 105 cells/well) were seeded into 6-well plates and treated with hBERA/miR-124-3p or LSA (15 nmol/L for A549, and 10 nmol/L for 143B and MG-63 cells), or vehicle. After 48 h, cells were detached with Cell Dissociation Buffer, washed with PBS, resuspended in RPMI medium containing Calcein AM (5 μmol/L), and then incubated at 37 °C for 30 min. Cells (1.0 × 105 cells/100 μL) were added to 96-well plates precoated with Collagen type I for attachment. To monitor cell adhesion capacity over time, cells were allowed to adhere for 1, 2, and 4 h at 37 °C, after which the experimental wells were aspirated and washed four times to remove non-adherent cells. The fluorescence intensity was measured using a SpectraMax® M3 microplate reader at an excitation of 494 nm and emission of 517 nm. The percentage of adhesion was determined by dividing the corrected fluorescence of adherent cells by the total corrected fluorescence of cells added to each well.
3
Quantification of Adhesion of Glioblastoma Stem Cells
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GSCs were grown on FN in a Vybrant™ cell adhesion assay kit (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions. FN (0 [PBS only], 1, 5 and 10 μg/mL) was coated overnight on a 96-well plate at 37°C. After pre-incubating cells with 5 μM of a fluorescent probe, calcein AM, for 30 min, wells were seeded with 10,000 cells of a calcein-labeled GSC suspension and cells incubated for another 2 h. Non-adhering cells were washed off twice with serum-free media. After adding 200 μL PBS to each well, a microplate reader was used to measure fluorescence at 490 nm (BioTek, Winooski, VT, USA). The amount of cells that adhered was measured from the fluorescence of adherent cells per well, after background fluorescence was subtracted, divided by that of added cells (after subtraction of background) multiplied by 100%.
4
Quantifying Cell Adhesion via Fluorescence
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Cell adhesion was measured using the Vybrant™ Cell Adhesion Assay Kit according to the manufacturer’s instructions (Thermo Fisher Scientific, Rockford, IL, USA). The cells, treated with ALA (0–10 mM) for 24 h, were loaded with calcein acetoxymethyl ester AM (5 µM) at 37 °C for 30 min. Calcein AM is non fluorescent, but, once loaded into cells, is cleaved by endogenous esterases to produce highly fluorescent calcein. Then, 100 μL of the calcein-labeled cell suspension was added to prepared microplate wells with ECM proteins, which was then incubated at 37 °C for 120 min. Non-adherent calcein-labeled cells were removed by careful washing, and the fluorescence was measured using a microplate reader (absorbance maximum of 494 nm and emission maximum of 517 nm, Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany).
5
Cell Adhesion Assay Using Calcein AM
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A cell adhesion assay was performed using the Vybrant Cell Adhesion Assay Kit (V-13181, ThermoFisher Scientific) according to the manufacturer’s protocol. Briefly, MSCs were stained with calcein AM solution (final concentration at 5 μM) by incubation at 37°C for 30 min, and 5000 cells were plated into prepared tissue culture plastic, collagen I-coated, or fibronectin-coated 96-well microplates. After incubation at 37 °C for 45 min to 4.5 h, nonadherent calcein-labeled cells were removed by careful washing, and 200 μl PBS was added to each well. The fluorescence intensity of each well was measured using a fluorescein filter set (494 nm absorbance maximum and 517 nm emission maximum). The total fluorescence intensity of plated cells was obtained by omitting the wash steps. The percentage of adhesion was determined by dividing the fluorescence of adherent cells by the total corrected fluorescence of cells added to each microplate well and multiplying by 100%.
6
Quantifying Endothelial Cell Adhesion to Collagen
7
Fluorescent Labeling for Co-Culture
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For co-culture experiments fibroblasts, HUVECs, and THP-1 cells were pre-labeled with calcein before the encapsulation in gelatin. Fibroblasts and HUVECs were stained with green calcein (Vybrant Cell Adhesion Assay Kit (V-13181), ThermoFisher Scientific), whereas THP-1 cells were stained with red-orange calcein [CellTrace™ Calcein Red-Orange, AM (C34851) ThermoFisher Scientific].
The fluorescent indicators, green and red calcein, were prepared as 1 mM stock in DMSO and stored at −20°C until use. Cells were centrifuged to get cell pellet and were resuspended in staining solution. Cells were labeled at a concentration of 1 × 106 cells mL−1 and a dye concentration of 5 μM (in serum free medium) of calcein for 30 min at 37°C in the dark and subsequently washed twice in PBS. Cells were resuspended in gelatin for encapsulation.
The fluorescent indicators, green and red calcein, were prepared as 1 mM stock in DMSO and stored at −20°C until use. Cells were centrifuged to get cell pellet and were resuspended in staining solution. Cells were labeled at a concentration of 1 × 106 cells mL−1 and a dye concentration of 5 μM (in serum free medium) of calcein for 30 min at 37°C in the dark and subsequently washed twice in PBS. Cells were resuspended in gelatin for encapsulation.
8
Monocyte Adhesion to Endothelial Cells
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The adhesion of monocytes (THP-1 cells) to EA.hy926 cells was determined using the Vybrant Cell Adhesion Assay Kit (ThermoFisher Scientific). EA.hy926 cells were seeded in 96-well flat bottom plates (density of 2 × 105 cells/mL, 1 × 105 cell per well). After incubation with FAs for 48 h and then with TNF-α for 24 h, calcein-labelled THP-1 cells (5 × 104 cells in 100 µL) were incubated with EA.hy926 cells for 1 h at 37 °C. Non-adherent THP-1 cells were removed by gentle washing, 100 µL PBS added to each well and co-cultures read on the Glomax Discover System (Promega). THP-1 monocyte adhesion was measured as a percentage of control (non-stimulated DMEM treated cells). Images of fluorescence-labelled THP-1 monocytes bound to EA.hy926 cells were taken with a Nikon Elipse Ti using NIS elements software (version 4.30).
9
Adhesion of Expanded CB-HSPCs
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Expanded CB-HSPCs were incubated with hiPSC-EVs for 2 h and stained with calcein AM (Vybrant Cell Adhesion Assay kit; Thermo Fisher Scientific), according to the manufacturer’s protocol. Next, cells were seeded onto 96-well culture plates either coated with fibronectin (50 µg/ml, Sigma-Aldrich) or covered with the monolayer of hMSCs or HUVECs. Unattached cells were washed out after 2.5 h and the fluorescent signal from remaining CB-HSPCs was measured by Infinite M200 Pro analyzer (Tecan, Männedorf, Switzerland). In addition, images of the attached cells were captured using fluorescent microscopy.
10
Monocyte Adhesion to Endothelial Cells
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The adhesion of THP-1 monocytes to the EA.hy926 cells was determined using the Vybrant Cell Adhesion Assay Kit (ThermoFisher Scientific, Waltham, MA, USA). The EA.hy926 cells were seeded in 96-well flat-bottomed plates (Corning, Corning, NY, USA) (cell density of 2 × 105 cells/mL, 1 × 105 cells per well). After incubation with FAs for 48 h, ECs were incubated with DMEM for 6 h. Calcein-labelled THP-1 cells (5 × 104 cells in 100 µL) were then incubated with the EA.hy926 cells for 1 h at 37 °C. The co-cultures were carried out in high glucose DMEM supplemented with 10% fetal bovine serum and 1% L-glutamine-penicillin-streptomycin solution. Non-adherent THP-1 cells were removed by gentle washing and 100 µL phosphate-buffered saline (PBS) was added to each well and the co-cultures were read on the Glomax Discover System (Promega, Southampton, UK). THP-1 monocyte adhesion was measured as a percentage of control (ECs incubated with DMEM, and then with calcein-labelled THP-1 cells). Images of fluorescence-labelled THP-1 monocytes bound to the EA.hy926 cells were taken with a Nikon Elipse Ti using NIS elements software (version 4.30, Nikon Instruments, Amsterdam, The Netherlands).
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