Neutrophil elastase was detected using anti-human NE antisera raised in rabbits [24 (link)]. Approximately 3 x 105 cells were seeded onto Superfrost slides and treated as described previously [24 (link)]. Briefly, primary rabbit anti-peptide antibodies specific for each human protease were diluted 1/200 (anti-NE) and bound antibodies were detected with an anti-IgG coupled to FluoProbes®-488 (Interchim, Montluçon, France). DRAQ5™ (Interchim, Montluçon, France) (10 μM) was used to detect dsDNA. Samples were examined with an Olympus FV 500 confocal microscope (Olympus, Rungis, France). Purified peripheral blood neutrophils were activated by incubation with P. aeruginosa at a MOI (multiplicity of infection) of 1:20 for 1 h at room temperature before evaluation as described in Brea et al [24 (link)].
Draq5
Manufactured by Interchim
Sourced in France
DRAQ5™ is a fluorescent dye used for nucleic acid staining in live-cell and fixed-cell applications. It is a far-red fluorescent DNA dye that can be excited with a variety of laser sources and emits at a long wavelength, making it suitable for multicolor flow cytometry and microscopy experiments.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using draq5
1
Neutrophil Elastase Detection by Immunofluorescence
2
AAV8 Particle Quantification in HeLa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa cells were seeded in 8-well μ-Slide ibiTreat chambers (Biovalley, Nanterre, France) at 2 × 105 (for 1- or 5-hr infections) or 8 × 104 (for 16-hr infection) cells/well. The day after, cells were infected or not with the rAAV vectors at a multiplicity of 20,000 VG/cell in duplicate. After 1, 5, or 16 hr, cells were washed in PBS, fixed with 4% paraformaldehyde in PBS, and permeabilized with 0.2% Triton X-100 in PBS. Permeabilized cells were blocked with PBS containing 10% goat serum (Sigma-Aldrich), incubated with ADK8 antibody (undiluted hybridoma culture supernatant) recognizing AAV8 intact capsid,21 (link), 47 (link) and washed with PBS. Cells were then incubated with anti-mouse Alexa Fluor 555 secondary antibody (1:200 in PBS), washed with PBS, and incubated with DraQ5 (1:1,000 in PBS) for nuclei staining (Interchim, Montluçon, France). Slides were finally mounted with Prolong Gold antifade reagent (Life Technologies). Images were captured on a Nikon A1 confocal microscope (Nikon Instruments, Amsterdam, Netherlands), and quantification of AAV8 particles according to their localization was then performed using the compartmentalization task of Volocity software (PerkinElmer, Courtaboeuf, France). Images were processed with ImageJ software (https://imagej.nih.gov/ij/ ) for picture presentation.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!