in vitro adhesion assay to detect the adherence of cancer cells to neutrophil extracellular traps

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

Add 100 microliters of the NET stock into each well of a 96-well flat bottom plate, and incubate the plate overnight at four degrees Celsius in the dark to coat the wells. 12 to 20 hours later, use a microscope to verify formation of a uniform monolayer of cell-free NETs at the bottom of the wells. Once the monolayer has been verified, gently aspirate all non-adherent material out of the wells, making sure not to disrupt the monolayer at the bottom.
This is the most challenging aspect of this procedure. In order to maintain an adequate NET monolayer, you have to handle the plate very carefully. Add all reagents and cells slowly on the side of the wells. Aspirate very gently, and avoid touching the bottom of the well with the suction tip.
With the non-adherent material removed, gently add 100 microliters of a 1% bovine serum albumin blocking solution to each well, and leave for one hour at room temperature. Do not agitate or shake the plate as this can disrupt the NET monolayer.
Next, prepare A549 cancer cells by harvesting them when they are 70 to 80% confluent using standard techniques. Resuspend the cells in medium at a concentration of 2 x 104 cells per 100 microliters. Stain the cells by adding one microliter of CFSE per milliliter of medium, and leave them at room temperature for 10 minutes.
Next, centrifuge the cells at 450 times g at four degrees Celsius for five minutes. Discard the supernatant, and resuspend the cells in the initial volume of medium to maintain a concentration of 2 x 104 cancer cells per 100 microliters of medium.
Take the NET-coated plate, and gently aspirate the blocking solution. Then, add 100 microliters of the cancer cell suspension to each well, and allow the cells to adhere to the plate for 90 minutes at 37 degrees Celsius and 5% carbon dioxide.
Fully aspirate the liquid, and add 1,000 units of DNase I to some wells for 10 minutes to degrade the NETs. In other wells, add 100 microliters of sterile water per well for 10 minutes as a vehicle control. Next, gently aspirate the liquid from each well, and wash with 100 microliters of PBS to remove any non-adherent A549 cells.
Aspirate and discard all solution in the wells, leaving only the NETs and adherent cancer cells at the bottom. Then, add 100 microliters of a 4% formaldehyde solution per well to fix the cells. Immediately transfer the plate to a fluorescence microscope to read the assay, and then, plot and analyze the results using the data analysis software.

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1. NET-Cancer Cell Static Adhesion Assay
<ol>
	<li>Add 100 &#956;l of the previously obtained NET stock per well in a 96-well flat bottom plate and allow to coat wells O/N at 4 &#176;C in the dark.</li>
	<li>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.</li>
	<li>Add 100 &#956;l of 1% Bovine Serum Albumin (BSA) blocking solution to each well and leave for 1 h at RT.</li>
	<li>Detach A549 cancer cells from a T-75 flask using 2 ml of 0.25% Trypsin solution. Once detached, add 10 ml of A549 media to trypsinized cells and centrifuge at 450 x g at 4 &#176;C for 5 min. Discard supernatant and resuspend cells in media to obtain a concentration of 2 x104 cancer cells per 100 &#956;l media. 
	NOTE: A549 cells were grown separately. Briefly, cells were cultured and maintained in DMEM F12 media containing 10% FBS and 1% Penicillin Streptomycin and incubated at 37 &#176;C 5% CO2. Once 70 - 80% cell confluence was reached, they were detached using 0.25% Trypsin-EDTA and resuspended in the same media described above.</li>
	<li>Stain cancer cells using CFSE by adding 1 &#956;l of CFSE per ml of media and leaving it to stain at RT for 10 min. After staining, centrifuge down at 450 x g at 4 &#176;C for 5 min then discard supernatant and resuspend cells in the initial volume of media to obtain a concentration of 2 x104 cancer cells per 100 &#956;l media.</li>
	<li>After 1 hr of NETs blocking, gently aspirate the blocking solution and add 2 x104 cancer cells in media, which is equivalent to 100 &#956;l, per well over the NET monolayer and allow it to adhere for 90 min at 37 &#176;C 5% CO2. Gently aspirate the cells and add 100 &#956;l of PBS to each well to wash any non-adherent cells.</li>
	<li>In some wells, add 1000U of DNAse I per well for 10 min before washing to degrade NETs. This will serve as a negative control. In other wells, add 100 &#956;l of sterile water per well for 10 min, which will serve as the vehicle control (VC). 
	NOTE: 3 replicates per condition are usually performed to increase sample size.</li>
	<li>Aspirate and discard all solutions in the wells leaving only NETs and adherent cancer cells at the bottom. Add 100 &#956;l of 4% formaldehyde solution per well to fix adherent cancer cells to NETS and read the assay under the fluorescence microscope (Figure 1). Plot and analyze the results (Figure 2).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (PBS), Modified, without calcium chloride and magnesium chloride</td>
			<td>Wisent</td>
			<td>311-425-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 Medium with L-glutamine</td>
			<td>Wisent</td>
			<td>350-000-CL</td>
			<td>3% RPMI solution is made by supplementing RPMI with 3% Fetal Bovine serum</td>
		</tr>
		<tr>
			<td>BD Pharm Lyse Lysing buffer</td>
			<td>BD Biosciences</td>
			<td>555899</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse1</td>
			<td>Roche</td>
			<td>11284932001</td>
			<td></td>
		</tr>
		<tr>
			<td>F12 DMEM</td>
			<td>Wisent</td>
			<td>319-075-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Wisent</td>
			<td>080-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>CFSE</td>
			<td>Invitrogen</td>
			<td>C1157</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
	</tbody>
</table>

Najmeh, S., et al. <a href="https://app.jove.com/v/52687">Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling.</a> 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.

<img alt="Figure 1" src="/files/ftp_upload/21804/21804_Figure1.jpg" /> 
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 &#956;m.
<img alt="Figure 2" src="/files/ftp_u...

1. NET-Cancer Cell Static Adhesion Assay

	Add 100 &#956;l of the previously obtained NET stock per well in a 96-well flat bottom plate and allow to coat ...

In the cancer microenvironment, activated neutrophils release neutrophil extracellular traps, or NETs, composed of extracellular DNA&#160; fibers, histones, and granular proteins, which play a crucial role in cancer progression.
To study cancer cell-NET adhesion in vitro, add NET suspension to multi-well plate wells. Incubate for NET monolayer formation.
Post-incubation, remove unadhered NETs.
Add a protein solution to block free binding sites in the wells.
Pipette fluorescently labeled cancer cells over NET monolayers.
Specific surface receptors on cancer cells interact with NETs, facilitating cell adhesion.
Post-incubation, discard media from the negative control well.
Add deoxyribonuclease to degrade NETs, detaching the cells.
Remove solutions from negative control and test wells. Use buffer to remove non-adherent cells.
Fix the adherent cancer cells to NETs with formaldehyde.
Using a fluorescence microscope, visualize the fluorescent cancer cells adhered to NETs.
Test wells show significant cancer cell adhesion to NETs, while deoxyribonuclease-treated NETs exhibit reduced levels of cancer cell adhesion.

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Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

隔离和中性粒细胞的表征与抗肿瘤性质

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

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Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

一种评估和定量中性粒细胞外细胞陷阱形成的高通量检测

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...

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

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