Neutrophils were seeded on glass coverslips treated with 0.001% polylysine, allowed to settle, and treated with PMA (25 nM) or left unstimulated. Cells were fixed with 4% PFA, blocked (3% normal donkey serum, 3% cold water fish gelatin, 1% bovine serum albumin, and 0.05% Tween 20 in PBS) and incubated with primary antibodies antinuclear membrane (ab12365; Abcam); anti–H2A–H2B–DNA complex (Losman et al., 1992 (link)); anti-neutrophil elastase (in-house); anti–S. aureus (Biodesign), which were detected with secondary antibodies coupled to Cy2 or Cy3 (Dianova). Controls were done with isotype-matched controls. For DNA detection, DRAQ5 (shown) Bisbenzimide 33342 (Hoechst 33342), Sytox, and ToPro3 were used. Specimens were mounted in Mowiol and analyzed with a PlanApo 63×/1.32 NA objective on a confocal microscope (TCS-SP; Leica).
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Neutrophil Imaging and Characterization
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Anti dna
Anti elastase
Antibodies
Antinuclear antibodies
Bisbenzimide
Bovine serum albumin
Cells
Cold
Confocal microscope
Donkey
Fish
Gelatin
Hoechst 33342
Isotype
Membrane
Neutrophil
Neutrophil elastase
Polylysine
Serum
Tween 20
Corresponding Organization :
Other organizations : New York University, Core Laboratories (United States), Max Planck Institute for Infection Biology, Charité - Universitätsmedizin Berlin
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Protocol cited in 11 other protocols
Variable analysis
independent variables
- Treatment with PMA (25 nM)
- Neutrophil morphology and localization of nuclear membrane, H2A–H2B–DNA complex, neutrophil elastase, and S. aureus
- Neutrophils seeded on glass coverslips treated with 0.001% polylysine
- Cells fixed with 4% PFA
- Cells blocked with 3% normal donkey serum, 3% cold water fish gelatin, 1% bovine serum albumin, and 0.05% Tween 20 in PBS
- Primary antibodies: anti-nuclear membrane (ab12365; Abcam), anti–H2A–H2B–DNA complex (Losman et al., 1992), anti-neutrophil elastase (in-house), and anti–S. aureus (Biodesign)
- Secondary antibodies coupled to Cy2 or Cy3 (Dianova)
- DNA detection using DRAQ5, Bisbenzimide 33342 (Hoechst 33342), Sytox, and ToPro3
- Specimens mounted in Mowiol and analyzed with a confocal microscope (TCS-SP; Leica)
- Isotype-matched controls
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