We thank Dr. Evan Jellison and Ms. Li Zhu in the flow cytometry core at UConn Health for their assistance with flow cytometry, Dr. Lynn Puddington in the Department of Immunology at UConn Health for her support of the instruments, Ms. Slawa Gajewska and Dr. Paul Appleton in the clinical research core at UConn Health for their help in obtaining blood samples. We acknowledge Dr. Christopher &#34;Kit&#34; Bonin and Dr. Geneva Hargis from UConn School of Medicine for their help with scientific writing and editing of this manuscript. This research was supported by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute (R01HL145454), National Institute of General Medical Sciences (P20GM121176), USA, a Career Development Award from the American Heart Association (18CDA34110426), and a startup fund from UConn Health. Figure 1 was created with BioRender.com.

Heparinized whole-blood samples were obtained from de-identified healthy human donors after obtaining informed consent, as approved by the Institutional Review Board of UConn Health, following the principles of the Declaration of Helsinki. Informed consent was obtained from all donors. The inclusion/exclusion criteria for this study were carefully developed to ensure the suitability of participants and to minimize potential risks. Eligible participants were aged between 18 and 65 years, of any ethnicity, fluent in English, and capable of providing informed consent. Excluded participants included those unable to provide informed consent for themselves, such as those requiring a legally authorized representative, individuals under 18 or over 65 years, incarcerated individuals, and pregnant women. Additionally, participants had to be free from anti-inflammatory medication usage and inflammatory conditions. Current infections or ongoing chronic or acute inflammatory conditions were also exclusion criteria. Finally, individuals with a current or recent history of COVID-19 infection were ineligible for the study. These criteria were designed to ensure participant safety and suitability while minimizing potential confounding factors that could impact the study results.
1. Preparation of reagents
<ol>
	<li>Neutrophil medium: Prepare the neutrophil medium by adding 2% human serum albumin to RPMI-1640 without phenol red (see Table of Materials).</li>
	<li>fMLP solution: Prepare the fMLP solution by diluting it 100 times from the 10 mM fMLP dimethyl sulfoxide (DMSO) storage solution (see Table of Materials) with RPMI 1640 without phenol red, resulting in a 100 &#181;M fMLP solution.</li>
	<li>Antibody solution: Dilute 120 &#181;L of allophycocyanin (APC)-conjugated monoclonal antibody mAb24 (mAb24-APC) (see Table of Materials), which reports the high-affinity conformation of &#946;2 integrin, in 10 mL of neutrophil medium, creating a 1.2 &#181;g/mL mAb24-APC solution.</li>
	<li>Compound library: Dilute the initial compound library with a concentration of 10 mM to 1 mM by adding 5 &#181;L of the 10 mM library (see Table of Materials) to 45 &#181;L of DMSO in 384-well plates using the liquid handler. Subsequently, transfer 5 &#181;L per well of the 1 mM compound library to an empty 384-well plate using the liquid handler.</li>
	<li>As illustrated in Figure 1, four columns (1, 2, 23, 24) contained only DMSO but no compounds, serving as positive and negative controls in the screening. Further dilute each well by adding 45 &#181;L of RPMI-1640 without phenol red, resulting in a final concentration of 100 &#181;M for all compounds.</li>
</ol>
2. Neutrophil isolation from human blood
<ol>
	<li>Using a serological pipette, carefully layer 4 mL of blood over 8 mL of a commercially available density gradient medium (see Table of Materials) in a 15 mL centrifuge tube (Figure 2A).</li>
	<li>Centrifuge the blood at 550 &#215; g for 30-50 min at 20 &#176;C. Decelerate the rotor gradually (deceleration factor of 1). 
	NOTE: The successful separation of neutrophils (band 2 in Figure 2B) from red blood cells may vary among donors. For most donors, a 30 min centrifugation is sufficient. However, for some donors, an additional 10-30 min of centrifugation may be required if separation is not successful (Figure 2C).</li>
	<li>Carefully remove the plasma (the yellow liquid on top) and mononuclear cells (the upper cloudy band; PBMC band in Figure 2B) using a 1 mL pipette.</li>
	<li>Collect neutrophils from the lower cloudy band (neutrophil band in Figure 2B) and approximately 3-4 mL below the clear liquid into a 15 mL centrifuge tube containing 10 mL of phosphate-buffered saline (PBS). Gently mix the neutrophil suspension by inverting it 2-3 times.</li>
	<li>Centrifuge the suspension at 400 &#215; g for 10 min at 20 &#176;C, and then carefully remove the supernatant by decanting.</li>
	<li>Gently resuspend the pellet with 5 mL of PBS and centrifuge it at 300 &#215; g for 5 min at 20 &#176;C.</li>
	<li>Remove the supernatant by decanting, and carefully eliminate any residual supernatant around the tube mouth and on the tube wall using vacuum suction. Resuspend the pellet in 1 mL of neutrophil medium.</li>
	<li>Count the cell numbers using a hemocytometer. Typically, 1 to 4 &#215; 107 neutrophils were obtained from 8 mL of human blood. 
	NOTE: There may be red blood cell contamination in the neutrophil suspension. Red blood cells do not significantly affect most assays and can prevent neutrophil activation/priming27. Red blood cells need to be lysed before counting to obtain an accurate neutrophil concentration. Add 10 &#181;L of the cell suspension to 891 &#181;L of deionized water for 10-30 s to lyse the red blood cells, then add 99 &#181;L of 10&#215; PBS to balance the osmotic pressure, preventing lysis of the neutrophils.</li>
	<li>Adjust the cell density to 6.25 &#215; 105 cells/mL by adding neutrophil medium.</li>
</ol>
3. Preparation of the 384-well plate
<ol>
	<li>In a 1.5 mL centrifuge tube, combine 1 mL of the antibody solution (1.2 &#181;g/mL mAb24-APC) with 0.2 mL of neutrophil medium to create a 1 &#181;g/mL mAb24-APC solution. Then, add 25 &#181;L of this mixture to the negative control wells in a 384-well plate (the blue wells in Figure 1).</li>
	<li>In a 15 mL centrifuge tube, mix 9 mL of the antibody solution with 21.6 &#181;L of the fMLP solution (100 &#181;M) and 1.7784 mL of neutrophil medium to create a solution containing 1 &#181;g/mL mAb24-APC and 200 nM fMLP. Next, add 25 &#181;L of this mixture to the positive control and testing wells in a 384-well plate (the red and blue wells in Figure 1).</li>
	<li>Transfer 5 &#181;L of the 100 &#181;M compound solutions from the compound library plate to the 384-well plate using a multi-channel pipette.</li>
	<li>Centrifuge the liquid for 1 min at 500 &#215; g, at room temperature. 
	&#8203;NOTE: To centrifuge plates, a swing-bucket rotor and plate buckets are required.</li>
</ol>
4. Treatment of cells
<ol>
	<li>Add 20 &#181;L of the neutrophil suspension to each well using a 16-channel pipette (5-50 &#181;L range, for each column of the 384-well plate). Gently mix 5-10 times using the pipette, maintaining a consistent time interval between each pipetting. 
	NOTE: The final concentrations in the wells are as follows: neutrophils 2.5 &#215; 105 cells/mL (all wells); mAb24-APC 0.5 &#181;g/mL (all wells); fMLP 100 nM (positive control and testing wells); compounds 10 &#181;M (testing wells).</li>
	<li>Incubate the plate on a shaker at 300 rpm, at room temperature (RT), for 10 min.</li>
	<li>Fix the cells by adding 3 &#181;L of 16% paraformaldehyde (PFA) to each well using a 16-channel pipette (final PFA concentration ~0.91%). Then, incubate on ice for 10 min. 
	&#8203;NOTE: It is recommended to maintain a consistent time interval between different columns when adding neutrophils and PFA. This ensures that neutrophils in each well have the same incubation time with fMLP and mAb24-APC.</li>
</ol>
5. Flow cytometry
<ol>
	<li>Turn on the flow cytometer (see Table of Materials), and ensure that the computer connected to the instrument is also turned on.</li>
	<li>Ensure that the sheath fluid container is filled, and the waste container is empty and properly connected to the instrument.</li>
	<li>Load the 384-well plate onto the sample loader of the flow cytometer. Ensure that the A1 well of the plate aligns with the A1 mark on the loader.</li>
	<li>Open the software and create a new experiment. Select 384 well and choose the desired fluorophores. Next, click on Plate setup, drag to select all wells, and then click on Sample. Turn on the &#34;High throughput&#34; switch, and select High under the rate panel.
	<ol>
		<li>In the volume box, enter 50. Select samples in odd-numbered columns and then activate the &#34;Agitation&#34; switch. Finally, name the samples in the right panel.</li>
	</ol>
	</li>
	<li>Click on Plot and gate. Then, click on the Apply button (two arrows on a green-filled circle), and subsequently, click on the play button (triangle shape). Wait for a few seconds to observe some cells from the first well, and then click on the pause button.
	<ol>
		<li>Adjust the FSC and SSC voltages to ensure proper visualization of cells on the plot. Click on Acquisition, and then click on the play button (triangle shape) to start the assay.</li>
	</ol>
	</li>
	<li>The flow cytometer will sequentially sample ~50 &#181;L of 1000-3000 neutrophils from each well. It will take approximately 3 h to complete the entire 384-well plate.</li>
	<li>After completing all samples, export the .fcs files for the next step.</li>
</ol>
6. Data analysis
<ol>
	<li>Open the flow cytometry data analysis software (see Table of Materials). Select and drag the folder containing all .fcs files into the software window, then release to import the data into the software.</li>
	<li>Double-click on one sample, gate single neutrophils based on FSC and SSC28,29,30 in the popped-up window, as shown in Figure 3. Select the gated rows and drag them to the folder, then release to apply the gating to all samples in that folder.</li>
	<li>Calculate and export the APC median fluorescence intensity (MFI) of neutrophils from each well using the software&#39;s table editor function. Click on Add Column under the Edit tab. Select Median in the Statistic tab, choose the gated population neutrophils, and select APC as the parameter. Click on the OK button.</li>
	<li>Return to the &#34;Table editor&#34; tab, select All Samples in the &#34;Group&#34; tab, and press the Create table button. A new window will appear displaying the created table. Save it as a text, CSV, or Excel file.</li>
	<li>Open the exported table, calculate the mean value and standard deviation of the APC MFI for the positive and negative control samples (Figure 4).</li>
	<li>Calculate the Z&#39;-factor (Z&#39;) of the plate using the equation: 
	Z&#39; = 1 - 3(&#963;p + &#963;n)/(&#181;p - &#181;n), 
	where &#963;p and &#963;n are the standard deviations of the positive and negative controls, respectively, and &#181;p and &#181;n are the means of the positive and negative controls, respectively31. 
	NOTE: The Z&#39; factor indicates the separation of the positive and negative control distributions. A Z&#39; of 0 means no separation, while a Z&#39; of 0.5 indicates equal separation. As mentioned in a previous study31, Z&#39; &#62; 0.5 indicates an excellent assay for compound screening. A Z&#39; in the range of 0.5 to 0 is acceptable, but it may only identify strong hits in the assay, which will require further validation in secondary assays.</li>
	<li>Consider compounds as &#34;hits&#34; for screening when the APC MFI of compound-treated neutrophils is lower than three times the standard deviation of the positive control MFI subtracted from the positive control MFI (P = 0.0013, represented by the dotted line in Figure 4).</li>
</ol>

Screening of β2 Integrin Activation Inhibitors in Human Neutrophils

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	<li>Shimaoka, M., Salas, A., Yang, W., Weitz-Schmidt, G., Springer, T.A. Small molecule integrin antagonists that bind to the &#946;2 subunit I-like domain and activate signals in one direction and block them in the other. Immunity. 19 (3), 391-402 (2003).</li>
	<li>Liu, W. et al. Nexinhib20 Inhibits neutrophil adhesion and &#946;2 integrin activation by antagonizing Rac-1-Guanosine 5&#8242;-Triphosphate interaction. The Journal of Immunology. 209 (8), 1574-1585 (2022).</li>
	<li>Robinson, M. et al. Antibody against the Leu-CAM beta-chain (CD18) promotes both LFA-1-and CR3-dependent adhesion events. The Journal of Immunology. 148 (4), 1080-1085 (1992).</li>
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	<li>Luque, A. et al. Activated conformations of very late activation integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region(355-425) of the common &#946;1 chain. Journal of Biological Chemistry. 271 (19), 11067-11075 (1996).</li>
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</ol>

The authors declare no competing financial interest.

The initiation and termination of neutrophil stimulation and staining are determined by the addition of neutrophils and the fixative PFA. Therefore, ensuring the same time interval between pipetting neutrophils or PFA into each column is critical. This ensures that the stimulation and staining time of neutrophils from each well remains consistent. Due to the short lifespan of neutrophils, the entire experiment, from collecting blood from donors to completing flow cytometry, must be carried out on the same day. Neutrophil...

Many inflammatory diseases are characterized by the infiltration of neutrophils at the site of swelling or injury1. To infiltrate these tissues, neutrophils must complete the neutrophil recruitment cascade, which involves arrest to the endothelium, extravasation across the vessel wall, and recruitment into the tissue2. Circulating neutrophils need &#946;2 integrin activation to complete this cascade, especially for the arrest phase. Thus, integrin-inhibiting drugs that reduce neutrophil adhesion, extravasation, and recruitment may effectively treat inflammatory diseases3,4.
&#946;2 integrins have been targeted for inflammatory diseases before. Efalizumab, a monoclonal antibody directly targeting integrin &#945;L&#946;2, was developed to treat psoriasis5. However, efalizumab was withdrawn due to its lethal side effect - progressive multifocal leukoencephalopathy resulting from JC virus reactivation6,7. New anti-inflammatory integrin-based therapies should consider maintaining the anti-infection functions of leukocytes to minimize side effects. The side effects of efalizumab might be due to the prolonged circulation of monoclonal antibodies in the bloodstream, which could inhibit immune functions in the long term8. A recent study shows that efalizumab mediates &#945;L&#946;2 crosslinking and the unwanted internalization of &#945;4 integrins, providing an alternative explanation for the side effects9. Thus, short-lived, small-molecule antagonists might avoid this problem.
A high-throughput method to screen small-molecule &#946;2 integrin antagonists using human neutrophils is presented here. &#946;2 integrin activation requires conformational changes of the integrin ectodomain to gain access to and increase its binding affinity to its ligand. In the canonical switchblade model, the bent-closed integrin ectodomain first extends to an extended-closed conformation and then opens its headpiece to a fully activated extended-open conformation10,11,12,13. There is also an alternative pathway that starts from the bent-closed to bent-open and extended-open, eventually14,15,16,17,18,19. The conformation-specific antibody mAb24 binds to an epitope in the human &#946;2-I-like domain when the headpiece of the ectodomain is open20,21,22,23.
Here, mAb24-APC is used to determine whether the &#946;2 integrins are activated. To activate neutrophils and integrin, N-formylmethionyl-leucyl-phenylalanine (fMLP), a bacterial-derived short chemotactic peptide that can activate neutrophil &#946;2 integrins24, is used as a stimulus in this protocol. When fMLP binds to the Fpr1 on neutrophils, downstream signaling cascades involving G-proteins, phospholipase C&#946;, and phosphoinositide 3-kinase &#947; are activated. These signaling events ultimately result in integrin activation via the inside-out signaling pathway18,25. Besides small molecule antagonists that directly bind to &#946;2 integrins and prevent conformational changes of integrin activation26, compounds that can inhibit components in the &#946;2 integrin inside-out activation signaling pathway would also be detected with this method. Automated flow cytometers enable high-throughput screening. Identifying new antagonists may not only deepen our understanding of integrin physiology but also provide translational insight into integrin-based anti-inflammation therapy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16-channel pipettes</td>
			<td>Thermo</td>
			<td>4661090N</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>384-well plate</td>
			<td>Greiner</td>
			<td>784201</td>
			<td>Materials</td>
		</tr>
		<tr>
			<td>APC anti-human CD11a/CD18 (LFA-1) Antibody Clone: m24</td>
			<td>BioLegend</td>
			<td>363410</td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Bravo&#160;Automated Liquid Handling Platform&#160;</td>
			<td>Agilent</td>
			<td>16050-102</td>
			<td>384 multi-channel liquid handler</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Model 5810R</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>Becton, Dickinson &#38; Company</td>
			<td>NA</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Human Serum Albumin Solution (25%)</td>
			<td>GeminiBio</td>
			<td>800-120</td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Lifitegrast</td>
			<td>Thermofisher</td>
			<td>&#160;50-208-2121</td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Nexinhib20</td>
			<td>Tocris</td>
			<td>6089</td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>N-Formyl-Met-Leu-Phe (fMLP)</td>
			<td>Sigma</td>
			<td>F3506</td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16% solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Plate buckets</td>
			<td>Eppendorf</td>
			<td>UL155</td>
			<td>Accessory</td>
		</tr>
		<tr>
			<td>Plate shaker&#160;</td>
			<td>Fisher</td>
			<td>88-861-023</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>PolymorphPrep</td>
			<td>PROGEN</td>
			<td>1895 (previous 1114683)</td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Prestwick Chemical Library Compound Plates (10 mM)</td>
			<td>Prestwick Chemical Libraries</td>
			<td>Ver19_384</td>
			<td>1520 small molecules, 98% marketed approved drugs (FDA, EMA, JAN, and other agencies approved)</td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium, no phenol red</td>
			<td>Gibco</td>
			<td>11-835-030</td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Swing-bucket rotor&#160;</td>
			<td>Eppendorf</td>
			<td>A-4-62</td>
			<td>Rotor</td>
		</tr>
		<tr>
			<td>ZE5 Cell Analyzer</td>
			<td>Bio-Rad Laboratories</td>
			<td>Model ZE5</td>
			<td>Instrument</td>
		</tr>
	</tbody>
</table>

a flow cytometry-based high-throughput technique for screening integrin-inhibitory drugs

This protocol aims to establish a method for identifying small molecular antagonists of &#946;2 integrin activation, utilizing conformational-change-reporting antibodies and high-throughput flow cytometry. The method can also serve as a guide for other antibody-based high-throughput screening methods. &#946;2 integrins are leukocyte-specific adhesion molecules that are crucial in immune responses. Neutrophils rely on integrin activation to exit the bloodstream, not only to fight infections but also to be involved in multiple inflammatory diseases. Controlling &#946;2 integrin activation presents a viable approach for treating neutrophil-associated inflammatory diseases. In this protocol, a monoclonal antibody, mAb24, which specifically binds to the high-affinity headpiece of &#946;2 integrins, is utilized to quantify &#946;2 integrin activation on isolated primary human neutrophils. N-formylmethionyl-leucyl-phenylalanine (fMLP) is used as a stimulus to activate neutrophil &#946;2 integrins. A high-throughput flow cytometer capable of automatically running 384-well plate samples was used in this study. The effects of 320 chemicals on &#946;2 integrin inhibition are assessed within 3 h. Molecules that directly target &#946;2 integrins or target molecules in the G protein-coupled receptor-initiated integrin inside-out activation signaling pathway can be identified through this approach.

Data from a representative 384-well plate screening (Figure 4) revealed that negative controls had an MFI of mAb24-APC of 3236 &#177; 110, while positive controls had an MFI of mAb24-APC of 7588 &#177; 858. The Z&#39; factor for this plate is approximately 0.33, which is within an acceptable range31. However, Z&#39; requires further validation in secondary assays.
To normalize the data, all values were scaled to assign a maximum value of 1 ...

Watch this Scientific Journal Video about Development of a Method for Identifying Small Molecular Antagonists of Î²2 Integrin Activation at JoVE.com

Author Spotlight: Development of a Method for Identifying Small Molecular Antagonists of &#946;2 Integrin Activation 

This protocol describes a flow cytometry-based, high-throughput screening method to identify small-molecule drugs that inhibit &#946;2 integrin activation on human neutrophils.

A Flow Cytometry-Based High-Throughput Technique for Screening Integrin-Inhibitory Drugs

This protocol aims to establish a method for identifying small molecular antagonists of &#946;2 integrin activation, utilizing ...

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Research

JoVE Journal

Immunology and Infection

1.5K Views.  UConn Health. This protocol describes a flow cytometry-based, high-throughput screening method to identify small-molecule drugs that inhibit β2 integrin activation on human neutrophils.

Video: Author Spotlight: Development of a Method for Identifying Small Molecular Antagonists of β2 Integrin Activation 

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...