Neutrophil Extracellular Trap (NET) Induction

Neutrophils were isolated from freshly collected whole blood of healthy adults or adult mice using the EasySep Direct Human Neutrophil Isolation kit (19666, Stemcell Technologies) or EasySep Mouse Neutrophil Enrichment Kit (19762, Stemcell Technologies), respectively, with greater than 95% purity (63 (link)). Neutrophils were resuspended to a concentration of 1 × 10⁶ cells/mL in Medium 199. Platelets were purified as described previously (66 (link), 67 (link)), resuspended to 1 × 10⁸ cells/mL in Medium 199, and activated with 50 ng/mL convulxin (sc-202554, Santa Cruz Biotechnology) for 15 minutes. Activated platelets and neutrophils were incubated at a 100:1 ratio to induce NETs for 2.5 hours at 37°C in 5% CO₂/95% air. HMGB1 was blocked by 10 μg/mL BoxA (HM-014, HMGBiotech). Neutrophils were pretreated for 1 hour with either nNIF or SCR peptides. NET levels were measured using the aforementioned MPO-DNA ELISA.

Free full text: Click here

Denorme F., Portier I., Rustad J.L., Cody M.J., de Araujo C.V., Hoki C., Alexander M.D., Grandhi R., Dyer M.R., Neal M.D., Majersik J.J., Yost C.C, & Campbell R.A. (2022). Neutrophil extracellular traps regulate ischemic stroke brain injury. The Journal of Clinical Investigation, 132(10), e154225.

Publication 2022

EasySep Direct Human Neutrophil Isolation kit EasySep Mouse Neutrophil Enrichment Kit

Adult Blood Cells Convulxin Elisa Hmgb1 Human Isolation Mouse Neutrophils Peptides Platelets Stemcell

Corresponding Organization :

Other organizations : DMS Imaging (France), University of Utah, University of Pittsburgh

Top 5 similar protocols

Variable analysis

independent variables

Blockade of HMGB1 using BoxA (10 μg/mL)
Pretreatment of neutrophils with nNIF or SCR peptides

dependent variables

NET levels measured using MPO-DNA ELISA

control variables

Neutrophils isolated from freshly collected whole blood of healthy adults or adult mice
Neutrophils resuspended to a concentration of 1 × 10^6 cells/mL in Medium 199
Platelets purified and resuspended to 1 × 10^8 cells/mL in Medium 199
Activated platelets and neutrophils incubated at a 100:1 ratio for 2.5 hours at 37°C in 5% CO2/95% air

positive control

Activated platelets and neutrophils incubated at a 100:1 ratio to induce NETs

negative control

Neutrophils pretreated with SCR peptides

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.

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?

Revolutionizing how scientists
search and build protocols!