Name: a flow cytometry-based high-throughput technique for screening integrin-inhibitory drugs
Uploaded: 2024-02-02T12:00:00.000+00:00
Duration: 4 min 15 s
Description: Watch this Scientific Journal Video about Development of a Method for Identifying Small Molecular Antagonists of β2 Integrin Activation at JoVE.com

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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</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. Neutrophils are highly sensitive to temperature changes and can become activated when exposed to rapid temperature increases, such as transitioning from 4 &#176;C to room temperature or from room temperature to 37 &#176;C. Additionally, based on our previous experience, fMLP-induced neutrophil &#946;2 integrin activation does not occur when cells are stored on ice or at 4 &#176;C (data not shown). Therefore, before fixation, whole blood and neutrophils should be kept at room temperature or 20 &#176;C (during the isolation centrifugation step). Do not place whole blood and neutrophils on ice.
The staining of mAb24 in negative controls should yield very low results. If a high mAb24 staining level is observed in an experiment, please check the following: (1) whether there was a significant temperature change during the experiment before fixation; (2) whether there was fMLP or endotoxin contamination in the neutrophil medium; (3) whether sample handling was too aggressive, such as generating bubbles during pipetting and mixing.
The current protocol employs mAb24 to report the opening of the &#946;2 integrin headpiece. Theoretically, KIM127, a monoclonal antibody reporting the extension of &#946;2 integrins34,35, can be used in conjunction with mAb24 to comprehensively assess &#946;2 integrin conformation. However, the signal-to-noise ratio of KIM127 staining (1.5 to 2-fold) is not as favorable as that of mAb24 (5 to 10-fold), which typically does not provide a satisfactory Z&#39; factor in the 384-well plate assay. In the 96-well plate assay, samples can be washed before performing flow cytometry, reducing soluble antibody-derived background signals. Therefore, the KIM127-based assay can be conducted in the 96-well plate assay, which has lower throughput compared to the 384-well plate assay.
Since this method uses fluorescence intensity as a readout to assess the integrin inhibitory effect of drugs, some fluorescent drugs may interfere with the results. Additionally, toxic drugs that induce neutrophil death within the 10 min stimulation period will also appear to report integrin inhibition. Drugs that inhibit neutrophil degranulation will suppress overall &#946;2 integrin expression. These degranulation inhibitors will also be identified in our screening. Therefore, secondary screening with other controls is needed to confirm the inhibitory effects of the hits. Secondary screens are referenced as a means to both validate and determine the mechanism of action of hits. In addition to repeating the mAb24 tests, the total CD18 expression on the cell surface will be assessed using a pan anti-human CD18 antibody. The level of activated &#946;2 integrin, as measured by mAb24, will be normalized by the total CD18 expression. This will enable us to determine whether the agent&#39;s mechanism of action involves antagonizing integrin activation and/or inhibiting the expression of CD18 on the cell surface. A viability assay should also be performed in the secondary screen to exclude any toxic effects of the hits.
This protocol has limitations. First, mAb24 is capable of detecting the &#946;2 I-like domain only in cases where the integrin is in a high-affinity state. Therefore, it cannot identify &#945;/&#946; I-like allosteric antagonists such as lifitegrast through decreasing mAb24 binding. Lifitegrast induces more mAb24 binding32 and shows an abnormally increased MFI value with mAb24 (Figure 4). For such abnormal values, other assays may be needed to verify whether these hits are &#945;/&#946; I-like allosteric antagonists like lifitegrast. Second, the Z&#39; factor for this assay is suboptimal and could potentially be improved through the use of automatic pipetting and agitation. Unfortunately, our laboratory lacks the necessary equipment to test this hypothesis. Duplicating or triplicating assays will be helpful in identifying false positives and negatives in case the Z&#39; factor cannot be further improved with the methods above. Furthermore, it may be beneficial to extend the incubation time to identify more hits, as observed with the known &#946;2 integrin activation inhibitor Nexinhib20, which requires an hour of incubation to produce inhibitory effects. This study focused on identifying fast-acting agents. Researchers should note that they can modify the incubation time to suit their specific needs.
To our knowledge, this is the first high-throughput screening method for &#946;2 integrin antagonists. This approach could be used to identify small molecule compounds that directly bind to &#946;2 integrins and prevent conformational changes leading to intermediate/high-affinity integrin states, similar to antagonists without &#34;agonistic&#34; properties recently described for integrins &#945;IIb&#946;3 and &#945;4&#946;126. Neutrophils are critical in many inflammatory diseases, such as myocardial ischemia-reperfusion injury36, sepsis37, and autoimmune diseases38,39. Small molecule drugs may offer more flexibility in treating these diseases compared to antibody-based drugs. Hits from our screen could provide potential treatments for inflammatory diseases.
The current method is a fluorescent antibody-based, high-throughput screen. Since activation reporter antibodies are also available for &#946;140,41,42,43,44, &#945;IIb&#946;345,46, and &#945;L integrins47,48,49,50, this method can be extended to identifying antagonists for other integrins. A conformationally sensitive antibody like HUTS-21, which binds to the &#946;1 hybrid domain51,52,53, has been used in a high-throughput screen to identify very late antigen-4 (VLA-4, integrin &#945;4&#946;1) allosteric antagonists54. The present screening method can also be modified and extended to find drugs that inhibit or promote the expression of other surface receptors, such as compounds that increase cystic fibrosis transmembrane conductance regulator (CFTR) surface expression on cystic fibrosis (CF) cells. In CF, multiple mutations lead to CFTR misfolding, resulting in absent expression of CFTR on the cell membrane55. Small-molecule drugs have been shown to restore the CFTR expression56. For protocol modifications, it is necessary to increase the incubation time of drugs to several hours to allow protein expression changes to occur.

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><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&amp;nbsp;Automated Liquid Handling Platform&amp;nbsp;</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 &amp;amp; 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>&amp;nbsp;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&amp;nbsp;</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&amp;nbsp;</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 to the positive mean and a minimum value of 0 to the negative mean. The Z&#39; factor will undergo more rigorous validation in secondary assays. The cutoff for this plate is set at 0.41, which means that samples with a relative MFI lower than 0.41 will be considered as hits inhibiting fMLP-induced &#946;2 integrin activation in human neutrophils. No hits were identified from this plate.
To confirm the protocol&#39;s effectiveness, Nexinhib20, which inhibits &#946;2 integrin activation by antagonizing Rac-1 function, and lifitegrast, which antagonizes integrin &#945;L&#946;2 directly32,33, were used. However, incubation times were adjusted to one hour for Nexinhib20 and half an hour for lifitegrast. The resulting data from these experiments were normalized using the same scaling method described above. These data points were then combined with the plate results and analyzed collectively (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64401/64401fig01.jpg" /> 
Figure 1: Plate layout for compound screening. A schematic diagram illustrating the arrangement of screening compounds and controls in a 384-well plate. Negative control wells are depicted in blue (columns 1 and 23), and positive control wells are shown in red (columns 2 and 24). Testing wells are represented in beige (columns 3 to 22). Arrows indicate the sequence for reading the plate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64401/64401fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64401/64401fig02.jpg" /> 
Figure 2: Neutrophil separation using density gradient medium. Representative photos demonstrating the successful and unsuccessful separation of neutrophils using density gradient medium. (A) Initially, 4 mL of blood is layered onto 8 mL of density gradient medium. (B) After centrifugation, two cloudy bands should be visible: the upper band containing primarily peripheral blood mononuclear cells (PBMCs) and the lower band containing mostly neutrophils with some red blood cells (RBCs). Most RBCs are pelleted at the bottom. (C) An unsuccessful separation where RBCs are not pelleted, and the neutrophil band is not observed. Additional centrifugation (10-30 min) would be required to separate the neutrophil band. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64401/64401fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64401/64401fig03.jpg" /> 
Figure 3: Gating of neutrophils using FSC/SSC plots. Representative forward scatter (FSC) and side scatter (SSC) plots illustrating the gating strategy for identifying neutrophils. (A) Neutrophils are gated based on the area of forward scatter (FSC-A) and side scatter (SSC-A) recorded by the flow cytometer. (B) Single cells are further gated based on the width (FSC-W) and height (FSC-H) of forward scatter, and (C) the width (SSC-W) and height (SSC-H) of side scatter. The color scale represents cell density, transitioning from red to yellow, green, and blue as density decreases. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64401/64401fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64401/64401fig04.jpg" /> 
Figure 4: Screening results in a representative 384-well plate. The screening results from a representative 384-well plate, demonstrating a Z&#39; factor of 0.3. Negative and positive controls are indicated by blue and red dots, respectively. Testing samples treated with various compounds are represented as beige dots. The dashed line represents the mean fluorescence intensity (MFI) cut-off for identifying hits. None of the tested compounds were identified as &#946;2 integrin antagonists, as all tested compounds displayed MFI values above the cut-off line. Results from independent experiments testing known &#946;2 integrin antagonists with varying incubation times (Nexinhib20 for 1 h, lifitegrast for half an hour) are pooled for presentation in this figure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64401/64401fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Development of a Method for Identifying Small Molecular Antagonists of β2 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 ...

a-flow-cytometry-based-high-throughput-technique-for-screening

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

1.6K Views.  UConn Health. Ons onderzoek is gericht op neutrofiele integrine, en in deze studie willen we een methode ontwikkelen om kleine moleculaire antagonisten van bèta-2-integrine-activering te vinden. Wij geloven dat we met deze methode nieuwe antagonisten kunnen vinden die ons begrip van integrinefysiologie kunnen verdiepen en ook translationeel inzicht kunnen bieden in op integrine gebaseerde ontstekingsremmende therapie. In de toekomst zullen we ons richten op het ontdekken van nieuwe ontstekingsremmende gen...

Video: Auteur in de schijnwerpers: ontwikkeling van een methode voor het identificeren van kleine moleculaire antagonisten van β2-integrine-activering 

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

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 ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target protein. Due to its several advantages over monoclonal antibodies, aptamer development has gained enormous popularity among cancer researchers. Systematic evolution of ligands by exponential enrichment (SELEX) is the most common method of developing aptamers specific for proteins of interest. Following SELEX, a quick and efficient binding assay accelerates the process of identification, confirming the selectivity and specificity of the aptamer. 
This paper explains a step-by-step flow cytometric-based binding assay of an aptamer specific for epithelial cellular adhesion molecule (EpCAM). The transmembrane glycoprotein EpCAM is overexpressed in most carcinomas and plays roles in cancer initiation, progression, and metastasis. Therefore, it is a valuable candidate for targeted drug delivery to tumors. To evaluate the selectivity and specificity of the aptamer to the membrane-bound EpCAM, EpCAM-positive and -negative cells are required. Additionally, a non-binding EpCAM aptamer with a similar length and 2-dimensional (2D) structure to the EpCAM-binding aptamer is required. The binding assay includes different buffers (blocking buffer, wash buffer, incubation buffer, and FACS buffer) and incubation steps. 
The aptamer is incubated with the cell lines. Following the incubation and washing steps, the cells will be evaluated using a sensitive flow cytometry assay. Analysis of the results shows the binding of the EpCAM-specific aptamer to EpCAM-positive cells and not the EpCAM-negative cells. In EpCAM-positive cells, this is depicted as a band shift in the binding of the EpCAM aptamer to the right compared to the non-binding aptamer control. In EpCAM-negative cells, the corresponding bands of EpCAM-binding and -non-binding aptamers overlap. This demonstrates the selectivity and specificity of the EpCAM aptamer. While this protocol is focused on the EpCAM aptamer, the protocol is applicable to other published aptamers.

A necessary step in anticancer aptamer development is to test its binding to the target. We demonstrate a flow cytometric-based assay to study this binding, emphasizing the importance of including a negative control aptamer and cancer cells that are positive or negative for that particular protein.&#160;

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target ...

Cancer Research

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells ...

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 ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-&#945;4&#946;7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn&#39;s disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.
The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.
Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling ...

Biology

A Fluorescence-Based Assay Using Engineered Lymphocytes for Screening Immunomodulatory Compounds

Source: Fouda, A., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55199">A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules.</a> J. Vis. Exp., (2017).
This video demonstrates the screening of immunomodulatory compounds via fluorescent-based assay. Transgenic mouse-derived T lymphocytes with green fluorescent protein expression cassettes are treated with these compounds. The enhanced and reduced GFP signals enable the identification of the compounds&#39; stimulatory and inhibitory effects, respectively.

1. Preparation of Splenocyte Medium and Flow-cytometry Buffer

	Perform all steps under a 70% ethanol-cleaned biological hood.
	Remove 70 mL of pre-warmed ...

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

Samenvatting

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