An In Vitro Technique to Generate Human Neutrophil Extracellular Traps
Published: August 31, 2023
Abstract
Source: Najmeh, S., et al. Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling. J. Vis. Exp. (2015)
This video demonstrates an in vitro technique to generate and isolate neutrophil extracellular traps (NETs). Neutrophils are isolated from peripheral blood and stimulated with phorbol myristate acetate (PMA) to produce NETs. During incubation, the neutrophils undergo a process of cell death (NETosis) to release NETs comprising decondensed chromatin mixed with granular cytoplasmic proteins.
Protocol
All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board. 1. NETs Generation Stimulate neutrophils with 500 nM of PMA (per 30 ml of neutrophil solution) and incubate on a 150 x 25mm flat tissue culture dish with a 20 mm grid for 4 hr at 37 °C 5% CO2. This will allow NETosis. Af…
Declarações
The authors have nothing to disclose.
Materials
| Dulbecco's Phosphate-Buffered Saline (PBS), Modified, without calcium chloride and magnesium chloride | Wisent | 311-425-CL | |
| RPMI-1640 Medium with L-glutamine | Wisent | 350-000-CL | 3% RPMI solution is made by supplementing RPMI with 3% Fetal Bovine serum |
| Phorbol 12-myristate 13-acetate (PMA) | Sigma-Alrdrich | P8139 | |
| Fetal Bovine Serum (FBS) | Wisent | 080-150 | |
| Integrid tissue culture dish with 20mm grid (150 x 25mm) | Falcon | 353025 |
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An In Vitro Technique to Generate Human Neutrophil Extracellular Traps. J. Vis. Exp. (Pending Publication), e21565, doi: (2023).