MCF10A and MCF12A cells were cultured with 200 ng/ml purified SEMA7A or PBS vehicle control in adherent (tissue culture plastic) or forced suspension conditions (ultra-low attachment plates (Corning, Corning, NY, USA: #3473)). Cells were seeded at a density of 1000 cells/well in a 96-well plate plus media only wells as controls. Cell death was measured 24 h post seeding via luminescence using the Caspase Glo assay (Promega, Madison, WI, USA: #G8090). Annexin-V /7AAD staining (Biolegend, San Diego, CA, USA: #640930) and pAKTS473 (ThermoFisher: Waltham, MA, USA, #17-9715-42) were also used to confirm cell viability by flow cytometry. See flow cytometry methods for staining protocol. Function blocking antibodies for b1 (CD29- 9EG7; BD Biosciences: #550531) and a6 integrin (ThermoFisher: #14-0495-82) were used to disrupt integrin signaling in the presence of SEMA7A or as antibody alone controls to determine off-target effects. IgG controls were also used. 9EG7 was used at a concentration of 0.6 μg/ml while GoH3 was used at a concentration of 100 μg/mL for inhibition studies consistent with previous reports [29 (link), 30 (link)]. Cells were cultured with anti-integrin inhibitors at time of seeding.
Cd29 9eg7
Manufactured by BD
CD29-9EG7 is a laboratory reagent used in cellular and molecular biology research. It is a monoclonal antibody that specifically binds to the CD29 (Integrin beta-1) cell surface protein. The core function of this product is to enable the detection and analysis of cells expressing CD29 using techniques such as flow cytometry, immunohistochemistry, or cell sorting.
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2 protocols using cd29 9eg7
1
Integrin signaling in SEMA7A-induced cell death
2
SEMA7A-Induced Cell Death in Adherent and Suspension Cultures
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MCF10A and MCF12A cells were cultured with 200ng/ml purified SEMA7A or PBS vehicle control in adherent (tissue culture plastic) or forced suspension conditions (ultra-low attachment plates (Corning, Corning, NY, USA: #3473)). Cells were seeded at a density of 1000 cells/well in a 96-well plate plus media only wells as controls. Cell death was measured 24 hours post seeding via luminescence using the Caspase Glo assay (Promega, Madison, WI, USA: #G8090). Annexin-V /7AAD staining (Biolegend, San Diego, CA, USA: #640930) and pAKTS473 (ThermoFisher: Waltham, MA, USA, #17-9715-42) were also used to confirm cell viability by flow cytometry. See flow cytometry methods for staining protocol. Function blocking antibodies for b1 (CD29-9EG7; BD Biosciences: #550531) and a6 integrin (ThermoFisher: #14-0495-82) were used to disrupt integrin signaling in the presence of SEMA7A or as antibody alone controls to determine off-target effects. IgG controls were also used. 9EG7 was used at a concentration of 0.6ug/ml while GoH3 was used at a concentration of 100ug/mL for inhibition studies consistent with previous reports (29, 30) . Cells were cultured with anti-integrin inhibitors at time of seeding.
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