Neutrophils were seeded on coverslips coated with poly-L-lysine and activated by 100 nM PMA for 3 h at 37 °C. Subsequently, cells were fixed using a 4% paraformaldehyde solution. Neutrophils were labeled with Sytox Green (Thermo Fischer Scientific, MA, USA) and Hoechst 33,342 (Sigma-Aldrich, MO, USA). Imaging was performed on an LSM700 Laser Scanning Confocal Fluorescence microscope (Zeiss). In each sample, 3 regions of interest containing 100–200 cells were evaluated for NETs formation manually. Non-NETting neutrophils were defined as those with compact DNA stained with both nuclear dyes. NETing neutrophils were defined as those having diffuse DNA stained only with Sytox Green.
Lsm700 laser scanning confocal fluorescence microscope
Manufactured by Zeiss
The LSM700 is a laser scanning confocal fluorescence microscope manufactured by Zeiss. It is designed to provide high-resolution imaging of fluorescently labeled samples. The LSM700 utilizes a laser light source to excite the fluorophores within the sample, and a confocal detection system to capture the emitted fluorescence, allowing for optical sectioning and improved image quality.
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Lab products found in correlation
3 protocols using lsm700 laser scanning confocal fluorescence microscope
1
Visualizing Neutrophil Extracellular Traps
2
Quantifying NET Formation in Neutrophils
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Imaging was performed on an LSM700 Laser Scanning Confocal Fluorescence microscope (Zeiss) using a 20× objective. Five regions of interest were collected in each section, and image analysis was done by Image J software. Applying antibody staining NE and H3, neutrophils not forming NETs were defined as those exhibiting a high‐intensity signal with NE (green) but a low‐intensity signal with H3 (red). NET‐forming neutrophils were defined as those exhibiting high‐intensity NE (green) and H3 (red) signals, showing colocalization (yellow). The percentage of NETs was calculated as the ratio of NET‐forming neutrophils to the total number of neutrophils (NET‐forming and nonforming neutrophils).
3
Quantifying Neutrophil Extracellular Traps
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Following activation, the neutrophils were labeled with Sytox Green (Invitrogen) and Hoechst 33342 (Sigma‐Aldrich) nuclear dyes. Images were taken with an LSM700 Laser Scanning Confocal Fluorescence microscope (Zeiss) using a 20× objective. Three regions of interest containing 100–200 cells were evaluated for each sample and NET formations were counted manually. Neutrophils not forming NETs were defined as those with compact DNA stained with both dyes. NET‐forming neutrophils were defined as those having diffused DNA stained only with Sytox Green. The percentage of NETs was calculated as the ratio of NET‐forming neutrophils to the total number of neutrophils (NET‐forming and nonforming neutrophils).
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