In Vitro Adhesion Assay to Detect the Adherence of Cancer Cells to Neutrophil Extracellular Traps
Published: November 30, 2023
Abstract
Najmeh, S., et al. Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling. J. Vis. Exp. (2015)
In this video, we demonstrate an assay utilizing fluorescence microscopy to detect the adhesion of cancer cells to neutrophil extracellular traps (NETs). In the test wells, there is a notable increase in the number of fluorescently labeled cancer cells adhering to NETs, whereas wells treated with DNase exhibit reduced cancer cell adhesion to NETs.
Protocol
1. NET-Cancer Cell Static Adhesion Assay Add 100 μl of the previously obtained NET stock per well in a 96-well flat bottom plate and allow to coat wells O/N at 4 °C in the dark. Between 12 and 20 hr later, verify the formation of a uniform monolayer of cell-free NETs at the bottom of the wells under the microscope (Figure 1). At this point, gently aspirate all non-adherent material out of the wells, making sure not to disrupt the NET monolayer at the botto…
Representative Results
Figure 1. NET Monolayer. Light microscopy images of the 96-well plate wells coated with NETs show (A) a uniform monolayer of NETs throughout the well as opposed to (B) inadequate coating of the well with an interrupted layer of NETs. Scale bars represent 40 μm. <img alt="Figure 2" src="/files/ftp_u…
開示
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 |
| BD Pharm Lyse Lysing buffer | BD Biosciences | 555899 | |
| DNAse1 | Roche | 11284932001 | |
| F12 DMEM | Wisent | 319-075-CL | |
| Fetal Bovine Serum (FBS) | Wisent | 080-150 | |
| Penicillin/Streptomycin | Gibco | 15140-122 | |
| CFSE | Invitrogen | C1157 | |
| 0.25% Trypsin-EDTA | Gibco | 25200-056 |
