The surface phenotype of EA.hy926 endothelial cells was determined by flow cytometry. Cells were fixed as described above. Direct labelling was performed for the following markers: endoglin/CD105 (BD Biosciences, Le Pont de Claix, France), ICAM-1/CD54 (BD Biosciences, Le Pont de Claix, France), and Platelet endothelial cell adhesion molecule PECAM1/CD31 (BD Biosciences, Le Pont de Claix, France). The cells were incubated for 30 minutes with antibodies. In all experiments, background labelling was assessed using the relevant fluorochrome-conjugated mouse IgG isotype control (BD Biosciences, Le Pont de Claix, France). Cells were centrifuged at 300 g for 5 minutes at 22°C then washed in PBS. Data acquisition was performed using a Guava easyCyte HT Flow Cytometer (Merck Millipore, Molsheim, France) and analysis carried out using the Incyte program. At least 10,000 events were collected for each sample.
Fluorochrome conjugated mouse igg isotype control
Manufactured by BD
Sourced in France
Fluorochrome-conjugated mouse IgG isotype control is a laboratory reagent used as a control in flow cytometry experiments. It is a mouse immunoglobulin (IgG) conjugated to a fluorescent dye, which serves as a negative control to measure non-specific binding or background fluorescence.
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2 protocols using fluorochrome conjugated mouse igg isotype control
1
Endothelial Cell Surface Phenotyping
2
Endothelial Cell Surface Marker Expression
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The marker surface expression of EA.hy926 endothelial cells was determined by flow cytometry. Cells were fixed beforehand as described above. Direct labelling was performed for the following markers: endoglin-CD105 (BD Biosciences Cat# 563264, RRID:AB_2738104 ), ICAM-1-CD54 (BD Biosciences Cat# 740978, RRID:AB_2740602 ) and Platelet endothelial cell adhesion molecule PECAM1-CD31 (BD Biosciences Cat# 744077, RRID:AB_2741979 ). CD54 and CD31 were two principal adhesion molecules, which determine changes in endothelial permeability, activation and transendothelial leukocyte migration. The cells were incubated for 30 min with antibodies. In all of the experiments, background labelling was assessed using the relevant fluorochrome-conjugated mouse IgG isotype control (BD Biosciences, Le Pont de Claix, France). Cells were centrifuged at 300 g for 5 min at 22 °C and then washed in PBS. Data acquisition was performed using a Guava easyCyte HT Flow Cytometer (Merck Millipore, Molsheim, France) and the data were analysed using the Incyte programme. At least 10,000 events were collected for each sample.
Mycoplasma testing was performed to confirm the absence of contamination according to the recommended protocol (MycoStrip™–Mycoplasma Detection Kit # rep-mys-50 InvivoGen).
Mycoplasma testing was performed to confirm the absence of contamination according to the recommended protocol (MycoStrip™–Mycoplasma Detection Kit # rep-mys-50 InvivoGen).
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