In Vitro Adhesion Assay to Detect the Adherence of Cancer Cells to Neutrophil Extracellular Traps

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

1. 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.
2. 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 bottom.
3. Add 100 μl of 1% Bovine Serum Albumin (BSA) blocking solution to each well and leave for 1 h at RT.
4. Detach A549 cancer cel....

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 repr.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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