Integrin Expression Analysis by Flow Cytometry

Integrin expression was monitored by flow cytometry and Geometric Mean Fluorescence Intensity (GMFI) was determined and normalized against the GMFI obtained for isotype controls. Staining was performed in a p96 U-bottom plate (Thermo Scientific) Cells were fixed with 2% formaldehyde for 10 minutes and blocked for 30 minutes with a PBS buffer containing 1% BSA and 100 μg/ml human gamma globulin (Sigma). When required, the cells were permeabilized with 0.1% Triton X-100 in PBS buffer for 10 minutes before blocking. The following hybridoma-derived monoclonal mouse antibodies were used to detect protein expression: BEAR-1 (integrin α_M) (24 (link)), Ts1/11 (integrin α_L) (25 (link)), HC1/1 (integrin α_X) (26 (link)), BU15 mAb anti-CD11c (Immunotools), Ts2/16 (integrin β₁) (27 (link)), Lia2/3 (integrin β₂) (28 (link)), PAINS-10 (tetraspanin CD9) (29 (link)), Vj1/12 (CD59) (30 (link)) and 5A6 (tetraspanin CD81) (31 (link)). Cells were washed and stained with a donkey anti-mouse Alexa Fluor^® 488 antibody (Life Technologies), as per the manufacturer’s instructions. To determine total cellular F-actin, Phalloidin-Alexa Fluor^® 647 (Life Technologies) was used. When indicated, cells were treated with 1 µM jasplakinolide (Santa Cruz) 24 hours previous to completing the retinoic acid-induced differentiation.

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Torres-Gomez A., Fiyouzi T., Guerra-Espinosa C., Cardeñes B., Clares I., Toribio V., Reche P.A., Cabañas C, & Lafuente E.M. (2022). Expression of the phagocytic receptors αMβ2 and αXβ2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells. Frontiers in Immunology, 13, 951280.

Publication 2022

human gamma globulin Alexa Fluor® 488 antibody Phalloidin-Alexa Fluor® 647 jasplakinolide

Alexa fluor 488 Alexa fluor 647 Anti antibody Bear Buffer Cd59 Cells Donkey F actin Flow cytometry Fluorescence Formaldehyde Gamma globulin Human Hybridoma Integrin Isotype Jasplakinolide Mouse Pains Phalloidin Protein Retinoic acid Tetraspanin Triton x 100

Corresponding Organization : Universidad Complutense de Madrid

Other organizations : Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Científicas

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Variable analysis

independent variables

Treatment with 1 µM jasplakinolide 24 hours previous to completing the retinoic acid-induced differentiation

dependent variables

Integrin expression as monitored by flow cytometry and determined by Geometric Mean Fluorescence Intensity (GMFI), normalized against the GMFI obtained for isotype controls

control variables

Staining was performed in a p96 U-bottom plate (Thermo Scientific)
Cells were fixed with 2% formaldehyde for 10 minutes
Cells were blocked for 30 minutes with a PBS buffer containing 1% BSA and 100 μg/ml human gamma globulin (Sigma)
When required, the cells were permeabilized with 0.1% Triton X-100 in PBS buffer for 10 minutes before blocking
Monoclonal mouse antibodies used to detect protein expression: BEAR-1 (integrin αM), Ts1/11 (integrin αL), HC1/1 (integrin αX), BU15 mAb anti-CD11c, Ts2/16 (integrin β1), Lia2/3 (integrin β2), PAINS-10 (tetraspanin CD9), Vj1/12 (CD59), and 5A6 (tetraspanin CD81)
Cells were washed and stained with a donkey anti-mouse Alexa Fluor® 488 antibody (Life Technologies)
To determine total cellular F-actin, Phalloidin-Alexa Fluor® 647 (Life Technologies) was used

positive controls

Isotype controls for antibody staining

negative controls

Not explicitly mentioned

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