In vitro differentiated murine MDSC (1 x 106 cells) stained with Hoechst (20 μM, ThemorFisher) and calcein (1mg/ml diluted 1:2000, Invitrogen) were co-cultured with the indicated tumor cells (ANV5, 4T1 or derivatives), previously stained with orange-CMRA (1mg/mL, diluted 1:2000, Invitrogen) and Hoechst. The coculture was embedded at 103 cells/μL in hydrogels. After 48 h (for mouse cells) and 20 h (for human cells), images were captured by a confocal microscope LSM 800 laser-scanning (Carl Zeiss) with Plan-apochromat-63x. Images in Z were acquired with Zen 2.3 software (Carl Zeiss) and were visualized with Volocity 3D (RRID:SCR_002668, ThermoFisher Scientific). NET area and the percentage of NETs was calculated using the plugin DANA for ImageJ (developed by Dr. Miriam Shelef). At least 60 cells per experimental condition were analyzed (59 (link)). Experiments were repeated three times.
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Quantifying Neutrophil Extracellular Traps
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Calcein
Cells
Coculture
Cultured tumor cells
Derivatives
Human
Hydrogels
Laser scanning confocal microscope
Mdsc
Mouse
Nets
Corresponding Organization : Clinica Universidad de Navarra
Other organizations : Unité en Sciences Biologiques et Biotechnologies de Nantes, Nantes Université, Centre National de la Recherche Scientifique, Institut de Cancérologie de l'Ouest, Friedrich-Alexander-Universität Erlangen-Nürnberg, University of Sheffield
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Variable analysis
independent variables
- Co-culture of in vitro differentiated murine MDSC (1 x 10^6 cells) and the indicated tumor cells (ANV5, 4T1 or derivatives)
- NET area
- Percentage of NETs
- In vitro differentiated murine MDSC stained with Hoechst (20 μM) and calcein (1mg/ml diluted 1:2000)
- Tumor cells (ANV5, 4T1 or derivatives) stained with orange-CMRA (1mg/mL, diluted 1:2000) and Hoechst
- Coculture embedded at 10^3 cells/μL in hydrogels
- Images captured by a confocal microscope LSM 800 laser-scanning (Carl Zeiss) with Plan-apochromat-63x after 48 h (for mouse cells) and 20 h (for human cells)
- Images in Z acquired with Zen 2.3 software (Carl Zeiss) and visualized with Volocity 3D (RRID:SCR_002668, ThermoFisher Scientific)
- NET area and the percentage of NETs calculated using the plugin DANA for ImageJ (developed by Dr. Miriam Shelef)
- At least 60 cells per experimental condition analyzed
- Experiments repeated three times
- No positive or negative controls were explicitly mentioned.
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