Mesd-FLAG Release from Apoptotic HEK293 Cells

HEK293 cells in 6-well plates were transfected with Mesd-FLAG plasmid using jetPRIME reagents (Polyplus Transfection, Illkirch, France). After 48 h, cells were cultured in 293 SFM II medium (Life Technologies) and treated with or without etoposide (200 μM) for 16 h. The conditioned media of apoptotic and healthy cells with released Mesd-FLAG were collected and centrifuged at 200 × g for 10 min at room temperature. Supernatants were filtered through 0.2-μm syringe filters (Pall Life Sciences, Port Washing, NY, low-protein binding), concentrated with Amicon centrifugal filter units (10 kDa, Millipore), and analyzed by Western blot using anti-FLAG M2 monoclonal antibody (Sigma) (Ding et al., 2015 (link)).

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Chen X., Guo F., LeBlanc M.E., Ding Y., Zhang C., Shakya A, & Li W. (2016). Mesd Extrinsically Promotes Phagocytosis by Retinal Pigment Epithelial Cells: Mesd regulates RPE phagocytosis. Cell biology and toxicology, 32(4), 347-358.

Publication 2016

jetPRIME reagents 293 SFM II medium 0.2-μm syringe filters Amicon centrifugal filter units anti-FLAG M2 monoclonal antibody

Anti antibody Apoptotic Cells Conditioned media Cultured medium Etoposide Hek293 cells Plasmid Syringe Transfection Western blot

Corresponding Organization : University of Miami

Other organizations : The First Affiliated Hospital, Sun Yat-sen University, Zhongshan Hospital, Fudan University, Sun Yat-sen University, Jinan City People's Hospital, Shandong First Medical University

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Variable analysis

independent variables

Etoposide treatment (200 μM)

dependent variables

Release of Mesd-FLAG protein in the conditioned media of apoptotic and healthy cells

control variables

HEK293 cell line
6-well plate format
Transfection with Mesd-FLAG plasmid using jetPRIME reagents
Culture in 293 SFM II medium
Centrifugation at 200 × g for 10 min at room temperature
Filtration through 0.2-μm syringe filters
Concentration using Amicon centrifugal filter units (10 kDa)
Western blot analysis using anti-FLAG M2 monoclonal antibody

controls

Cells not treated with etoposide (healthy cells)

Annotations

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