An oxidative stress assay was utilized to measure neutrophil ROS production, as discussed previously [19 (link)]. Neutrophils were incubated with the fluorescent dye CMH(2)CFDA (Life Technologies, Eugene, OR, USA) in DPBS at room temperature for 10 min in the dark and added to biofilms or planktonic cells at 2 × 105 neutrophils/well. Phorbol myristate acetate (PMA, Sigma, St. Louis, MO, USA) at 100 nM was added in a subset of experiments as a positive control. Fluorescence (excitation 495 nm/emission 527 nm) was measured every 30 min for 3 h. Background fluorescence was measured for each C. albicans condition without neutrophils and subtracted from total fluorescence values prior to data analysis.
Protocol detail
Neutrophil ROS Production Assay
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Corresponding Organization : University of Wisconsin–Madison
Top 5 similar protocols
Protocol cited in 1 other protocol
Variable analysis
independent variables
- Incubation of neutrophils with the fluorescent dye CMH(2)CFDA
- Addition of biofilms or planktonic cells to the neutrophils
- Addition of phorbol myristate acetate (PMA) at 100 nM as a positive control
- Neutrophil ROS production
- Fluorescence (excitation 495 nm/emission 527 nm)
- Incubation time (10 min) at room temperature in the dark
- Neutrophil density (2 × 10^5 neutrophils/well)
- Background fluorescence measurement for each C. albicans condition without neutrophils
- Phorbol myristate acetate (PMA) at 100 nM
- Fluorescence measurement for each C. albicans condition without neutrophils
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
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!