The authors acknowledge the following funding agencies: FAPDF, CNPq, CAPES, UnB, FINEP, and FINATEC.

All experiments strictly followed the ethical guidelines set by the institutional review board at the University of Brasilia (process 13364819.0.0000.5558), and samples were identified by codes to ensure donor anonymity. The cells were obtained from normal healthy male donors aged 18-35 years, who signed the informed consent and met the following eligibility criteria: non-smokers/vapers, no chronic health conditions, and no history of inflammatory conditions in the last 14 days.
1. Blood...

Neutrophil Functional Assays for Screening Different Signaling Pathways

The authors declare no conflict of interest.

Neutrophils are highly dynamic and responsive cells that are short-lived and cannot yet be cryopreserved19, making investigations into their biology challenging. Therefore, it is essential to follow careful steps to obtain viable, enriched, and resting neutrophils11,20. This study employed a density-based isolation technique that emphasizes gentle and minimal manipulation, as well as the use of low temperatures until the activation step. A...

Neutrophils are the most abundant innate immune cells in human blood and are known to play a major part in infection and inflammation, being the first responders to arrive at the site of tissue damage1. In recent years, there has been a growing recognition of the crucial role that neutrophils play in a variety of diseases and in supporting homeostasis2. Neutrophils not only have tightly regulated signaling themselves, but also participate in the regulation of other components of the immune system3,4,5. Therefore, investigating neutrophils and their many unusual cellular functions, such as chemotaxis, reverse migration6, phagocytosis7, respiratory burst8, and the release of neutrophil extracellular traps (NETs)7, is imperative in numerous research contexts where it is necessary to assess the potential neutrophil functional, morphological, or molecular changes triggered by specific conditions under analysis.
Freshly isolated neutrophils are terminally differentiated, short-lived, highly dynamic, and easily activated9. However, an efficient storage method that does not affect the neutrophil responses has not yet been achieved, making it challenging to perform multiple assays that must be uninterrupted. Furthermore, previously described functional analyses10,11, based on assays that require cytometry and/or fluorescent staining, may not be a viable choice when a broad and initial evaluation of the neutrophil is needed.
To address these issues, this protocol describes a set of tests that can be carried out following a single isolation process, including the evaluation of cell viability, reactive oxygen species (ROS) production, real-time migration, and phagocytosis of Saccharomyces cerevisiae, whose results can be used to reason in-depth follow-up studies. This procedure, named NeutroFun Screen, was designed to encompass the leading effector activities, except degranulation, and can be completed in an average time of 4 h, including 1 h of activation. Additionally, the remaining cells can be used for more detailed approaches like omics studies. The advantage of this method lies in its balance between speed, comprehensiveness, cost, and accuracy.
Furthermore, there is a way to easily observe a preliminary suggestion of NETs, without specific markers, but enough to indicate if further efforts in that direction would be worthwhile. The diversity of functions tested aims to combine common points among tests, reducing the analysis time and expenses. The main goal of this method is to provide a balanced, functional analysis regarding speed, comprehensiveness, cost, and accuracy that allows for an overview of the neutrophil&#39;s response, making it a useful initial step in investigating the effects of novel stimuli on normal density neutrophils.

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			<td>CIM-Plate 16</td>
			<td>Agilent&#160;</td>
			<td>5665825001</td>
			<td></td>
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			<td>CLARIOstar Plate Reader&#160;</td>
			<td>BMG LABTECH</td>
			<td></td>
			<td>US Patent Number 9,733,124 
			Product details: MARS Data Analysis Software</td>
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			<td>Dimethyl sulfoxide</td>
			<td>Din&#226;mica</td>
			<td>1582</td>
			<td></td>
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			<td>DNAse I</td>
			<td>Sigma - Aldrich</td>
			<td>DN 25</td>
			<td></td>
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			<td>Ethylenediaminetetraacetic acid disodium salt dihydrate</td>
			<td>Sigma - Aldrich</td>
			<td>E5134</td>
			<td></td>
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			<td>Fast panoptic stain</td>
			<td>Laborclin</td>
			<td>620529</td>
			<td></td>
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			<td>Glass slide</td>
			<td>Exacta</td>
			<td>7102</td>
			<td></td>
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			<td>Hank&#8217;s Balanced Salt Solution with calcium, with magnesium, without phenol red.</td>
			<td>Sigma - Aldrich</td>
			<td>55037C</td>
			<td></td>
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			<td>Hank&#8217;s Balanced Salt Solution without calcium chloride, magnesium sulfate and sodium bicarbonate.</td>
			<td>Sigma - Aldrich</td>
			<td>H4641</td>
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			<td>Heparin</td>
			<td>Blau</td>
			<td>&#160;7896014655229</td>
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			<td>Laminar flow cabinet</td>
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			<td>VLFS-12</td>
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			<td>Microscope</td>
			<td>Zeiss</td>
			<td>415501-0101-002</td>
			<td>Product details: Primostar 1</td>
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			<td>Mixing Block</td>
			<td>BIOER</td>
			<td>MB-102</td>
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			<td>Neubauer improved bright-lined</td>
			<td>New Optik</td>
			<td>1110000</td>
			<td></td>
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			<td>N-formyl-methionyl-leucyl-phenylalanine</td>
			<td>Sigma - Aldrich</td>
			<td>F3506</td>
			<td></td>
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		<tr>
			<td>Nitroblue tetrazolium</td>
			<td>Neon</td>
			<td>CAS 298-83-9</td>
			<td></td>
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			<td>Percoll</td>
			<td>Cytiva</td>
			<td>17089101</td>
			<td>separation media</td>
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			<td>Phorbol 12-myristate 13-acetate</td>
			<td>Sigma - Aldrich</td>
			<td>P8139</td>
			<td></td>
		</tr>
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			<td>Phosphate buffered saline tablet</td>
			<td>Sigma - Aldrich</td>
			<td>P4417</td>
			<td></td>
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			<td>ROTOFIX 32 A</td>
			<td>Hettich</td>
			<td>1206</td>
			<td></td>
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			<td>Saccharomyces cerevisiae</td>
			<td>Fleischmann</td>
			<td></td>
			<td></td>
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			<td>Safranin</td>
			<td>Sigma - Aldrich</td>
			<td>50240</td>
			<td></td>
		</tr>
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			<td>Sodium dodecyl sulfate</td>
			<td>Cytiva</td>
			<td>17-1313-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Qsonica</td>
			<td>Q125</td>
			<td></td>
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			<td>Trypan blue solution</td>
			<td>Vetec</td>
			<td>C.I. 23850</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Genie 2</td>
			<td>Scientific Industries, Inc.</td>
			<td>0K-0500-902</td>
			<td></td>
		</tr>
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			<td>xCELLigence Real-Time Cell Analysis (RTCA) DP (dual purpose)</td>
			<td>Agilent&#160;</td>
			<td>380601050</td>
			<td>Product details: RTCA system composed of detection hardware, cell plates and software</td>
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a set of screening techniques for a quick overview of the neutrophil function

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as chemotaxis, reverse migration, phagocytosis, degranulation of cytotoxic enzymes and metabolites, and release of DNA as neutrophil extracellular traps (NETs). Neutrophils not only have tightly regulated signaling themselves, but also participate in the regulation of other components of the immune system. As fresh neutrophils are terminally differentiated, short-lived, and highly variable among individuals, it is important to make the most of the collected samples. Researchers often need to perform screening assays to assess an overview of the many neutrophil functions that may be affected by specific conditions under evaluation. A set of tests following a single isolation process of normal density neutrophils was developed to address this need, seeking a balance between speed, comprehensiveness, cost, and accuracy. The results can be used to reason and guide in-depth follow-up studies. This procedure can be carried out in an average time of 4 h and includes the evaluation of cell viability, reactive oxygen species (ROS) production, real-time migration, and phagocytosis of yeast on glass slides, leaving enough cells for more detailed approaches like omics studies. Moreover, the procedure includes a way to easily observe a preliminary suggestion of NETs after fast panoptic staining observed by light microscopy, with a lack of specific markers, albeit enough to indicate if further efforts in that way would be worthwhile. The diversity of functions tested combines common points among tests, reducing the analysis time and expenses. The procedure was named NeutroFun Screen, and although having&#160;limitations, it balances the aforementioned factors. Furthermore, the aim of this work is not a definite test set, but rather a guideline that can easily be adjusted to each lab&#39;s resources and demands.

Author Spotlight: Balancing Speed, Cost, and Accuracy in Neutrophil Function Assessment with NeutroFun Screen Protocol 

A Set of Screening Techniques for a Quick Overview of the Neutrophil Function

The NeutroFun Screen Protocol provides a group of functional tests that use low-cost chemicals and can be performed in a single experiment from fresh neutrophils. This allows a preliminary evaluation of some major neutrophil functions to guide more laborious and expensive follow-up studies. Neutrophil research employs various modulation conditions.Such as cytokines, peptides, microbial products, and physical stimuli. Additionally, analyses flow cytometry, multiomics, microfluidic devices and immuno cytochemistry. The extensive brand of conditions and assays used in combination makes the workflow of neutrophil research long, laborious and expensive.Typically in these reduces, the number of conditions tested or the number of tests performed, the current state of the art methods require extensive configurables, many cells and lengthy preparation procedures. Our protocol NeutroFun Screen offers a quicker and more cost-effective approach to evaluate cell functionality compared to other methods. This is achieved by combining various analyses within the protocol, while maintaining accuracy and comprehensiveness in the results.Two researchers performed this workflow simultaneously for a quick overview of the neutrophil function. Researcher one isolates the PMNs, adjusts concentration and incubates the activation systems, whereas researcher two prepares materials for the subsequent cases. Then researcher one performs phagocytosis, and colorimetric NBT assays while researcher two conducts real-time migration and NBT slide tests.

The density-based isolation method used in this study (Figure 1) met the criteria for the proposed experiments. Neutrophil parameters obtained from this method included viability &#8805;98%, purity &#8805;94%, and cell yield &#8805;1.5 x 107, with no activation detectable&#160;by the screening tests. Two relevant steps in the isolation of PMNs are anticoagulation and RBC removal. Keeping the anticoagulated blood tube or syringe at a gentle rocking before layering over the density ...

Watch this Scientific Journal Video about Balancing Speed, Cost, and Accuracy in Neutrophil Function Assessment with NeutroFun Screen Protocol at JoVE.com

This protocol features a set of neutrophil functional assays to be used as a screening method to cover functions from different signaling pathways. The protocol includes an initial and simple evaluation of cell viability, purity, reactive oxygen species production, real-time migration, phagocytosis, and a preliminary suggestion of neutrophil extracellular traps.

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as ...

a-set-screening-techniques-for-quick-overview-neutrophil

Research

JoVE Journal

Immunology and Infection

Neutrophil Isolation and Activation

Nitrotetrazolium Blue Chloride (NBT) Assay for Evaluating ROS Production

Neutrophil Phagocytosis Assay

Real-Time Polymorphonuclear Neutrophils (PMN) Chemotaxis Assay

Neutrophil Extracellular Trap (NET) Suggestive Assay

1.2K Views.  University of Brasilia.  This protocol features a set of neutrophil functional assays to be used as a screening method to cover functions from different signaling pathways. The protocol includes an initial and simple evaluation of cell viability, purity, reactive oxygen species production, real-time migration, phagocytosis, and a preliminary suggestion of neutrophil extracellular traps.

Neutrophil Functional Assays for Screening Different Signaling Pathways

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