Name: a fluorescence-based lymphocyte assay suitable for high-throughput screening of small molecules
Uploaded: 2017-03-10T14:00:00
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This work was supported by the Merck Frosst Start-up funds provided by Universit&#233; de Montr&#233;al. We would like to thank Drs Jean Duchaine and Dominic Salois from the High-throughput platform at the Institute for Research in Immunology and Cancer for their discussion, comments and feedbacks. Moutih Rafei holds a Fonds de la Recherche en Sant&#233; du Qu&#233;bec Junior 1 Award.

All animal protocols were approved by the Animal Care Committee of Universit&#233; de Montr&#233;al. Mice were euthanized by gradual inhalation of CO2 until no vital signs were observed followed by cervical dislocation. The procedure was carried out by a certified person to ensure that animals were euthanized in a humane manner and according to the recommendations of the Canadian Council on Animal Care.
1. Preparation of Splenocyte Medium and Flow-cytometry Buffer
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	<li>Perfor...

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Several read-out methods have been exploited for the development of sensitive and reliable HTS assays. These include colorimetric, luminescent or fluorescent methods. Although colorimetric methods are simple to set-up, they require multiple additions of chemicals, which may interfere or disrupt the cells being tested.23 In addition, they do not permit dynamic assessment of a biological response as the pharmacological effect is assessed at a specific endpoint. Furthermore, this method may require e...

High throughput screening (HTS) is a proven strategy widely adopted for the identification of new therapeutic molecules or for the repositioning of FDA-approved drugs in new medical indications.1 So far, the achieved HTS success can be measured by the plethora of previously discovered drugs. For instance, the tyrosine kinase inhibitor lapatinib used for the treatment of breast cancer, sitagliptin; a dipeptidyl peptidase-4 (DPP-4) inhibitor used as an anti-hyperglycemic drug, and the oral Bcr-Abl tyrosine kinase inhibitor dasatinib used for the treatment of chronic myelogenous leukemia represent few examples of a long list of approved drugs originally discovered by HTS.2 Although the productivity of the pharmaceutical industry has lately suffered from a lack in the discovery of new chemical entities, the likelihood of successful drug discovery can be improved through an increase in the number of pre-clinical candidates displaying modulatory biological/biochemical properties. Accordingly, the development of new HTS assays adapted for phenotypic screening could offer the potential to provide important pharmacological tools for the discovery of new drug hits.3,4,5,6 Furthermore, HTS can now be performed at a faster pace due to significant technological transformations in recent years including custom-designed flexible robotic installations, novel read-out technologies and extensive miniaturization.2,7 Among the factors contributing to the growing interest in the use of phenotypic screening (aka forward pharmacology) is the perception that focusing on functional effects rather than oversimplified reductionist assumptions regarding molecular targets (target-based screening/biochemical reactions) is more likely to show clinical efficacy. Thus, phenotypic screening holds the promise to uncover new potentially therapeutic compounds and molecular pathways of currently untreatable diseases.2
To properly identify inhibitors or activators for a given molecular target or cellular function, a highly sensitive and reliable assay is required in order to differentiate between bona fide hits and false positives. So, what makes a good assay? The quality of a given assay must be first judged by the signal-to-noise ratio (reflected through a Z factor).8 Second, the targeted effect or the goal of the screen should be clearly established. For example, functional cell-based approaches can offer significant advantages for receptor screening as opposed to an assay specifically designed to assess ligand-receptor binding. The reason for this is that the latter approach cannot differentiate between agonist and antagonist ligands.9 In contrast, a cell-based approach is likely to be more effective as receptor function can be directly assessed in a biological phenotype (proliferation, cell cycle arrest, apoptosis, and/or differentiation). However, it must be noted that biochemical assays can provide significant advantages over phenotypic assays as they infringe on a specific intracellular target. A well-optimized biochemical assay will generally have less data scatter than a phenotypic screening while simplifying afterwards investigations related to the drug molecular mechanism of action. However, the major drawback of target-based or biochemical assays is the chance of amplifying the rate of false positive hits that may affect non-specific targets when tested in a biological system (loss of the specificity originally studied in the biochemical assay).10 Although a well-established cut-off point between negative and positive hits can minimize the number of false positives in the primary screening, the use of a physiologically relevant system mimicking the native cellular environment such as intact cells, whole tissue or whole animal remains the core of the assay design pendulum. Therefore, phenotypic screening enables lead discovery with desirable biological/phenotypic effects for diseases with no identified drug targets without having prior knowledge of the compound&#39;s activity or mode of action.11
The herein study concerns the development and testing of an optimized and reproducible phenotypic screening based on two important components: a commercially available mouse model and a clustered sub-family of chemical compounds. With respect to the animal model, the assay relies on the use of lymphocytes derived from a mouse strain (Nur77GFP) harboring a bacterial artificial chromosome containing a cassette in which the expression of the green fluorescent protein (GFP) is driven by the Nur77 promoter.12 The hallmark of this stimulation is based on the fact that Nur77 is an immediate early gene up-regulated following T-cell receptor (TCR) or B-cell receptor (BCR) stimulation.12 As for the screening method itself, an approach was used to help avoiding the screening of trivial analogues while minimizing the time needed to assess a large chemical library (&#62;105 compounds). To do so, a database of chemical compounds selected by medicinal chemists using virtual screening tools was exploited to identify topologically similar compounds using known active seed structures as references. This approach allowed us to screen 4,398 compounds representing an overall library of over 136,000 chemical entities.

<table border="0" cellpadding="0" cellspacing="0" width="725">
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:211px;">Nur77GFP mice</td>
			<td style="width:157px;">The Jackson Laboratory</td>
			<td style="width:135px;">Mouse strain No. 016617</td>
			<td style="width:224px;">An in house colony was established at our animal facility&#160;</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">96 wells-U culture plates, sterile</td>
			<td style="width:157px;">VWR International</td>
			<td style="width:135px;">10062-902</td>
			<td style="width:224px;">T-cell activation using the magnetic beads</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:211px;">70&#181;m cell strainer, sterile</td>
			<td style="width:157px;">Corning Inc.</td>
			<td style="width:135px;">352350</td>
			<td style="width:224px;">Generation of splenocytes cell suspension</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:211px;">5 ml polystyrene round bottom tubes, sterile</td>
			<td style="width:157px;">Corning Inc.</td>
			<td style="width:135px;">352058</td>
			<td style="width:224px;">Generation of splenocytes cell suspension</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:211px;">50 ml polypropylene conical bottom tubes, sterile</td>
			<td style="width:157px;">VWR International</td>
			<td style="width:135px;">89039-656&#160;</td>
			<td style="width:224px;">Generation of splenocytes cell suspension</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:211px;">10 ml syringe without needle, sterile</td>
			<td style="width:157px;">Becton, Dickinson and Company</td>
			<td style="width:135px;">305482</td>
			<td style="width:224px;">To mash the spleen</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">T-25 culture flask</td>
			<td style="width:157px;">Greiner Bio-One</td>
			<td style="width:135px;">690 175</td>
			<td style="width:224px;">To incudabte B cells during activation</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:211px;">5 ml cell culture dish, sterile</td>
			<td style="width:157px;">Greiner Bio-One</td>
			<td style="width:135px;">627 160</td>
			<td style="width:224px;">To mash the spleen</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">Penicillin- Streptomycin (10,000 U/mL)</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">450-200-EL&#160;&#160;</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:211px;">RPMI 1600 with sodium bicarbonate and L- glutamine</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">350-002-CL&#160;</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:211px;">MEM non-essential amino acids</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">321-010-EL&#160;</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">HEPES free acid 1 M</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">330-050-EL&#160;</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Sodium pyruvate solution (100mM)</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">600-110-EL&#160;</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="72">
			<td height="72" style="height:72px;width:211px;">Fetal Bovine Serum (FBS)&#160;</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">080-910&#160;</td>
			<td style="width:224px;">Component of the splenocyte media and flow-cytometry buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Phosphate buffered saline (PBS)</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">311-010-CL</td>
			<td style="width:224px;">Component of flow-cytometry buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">2-Mercaptoethanol (55mM)</td>
			<td style="width:157px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">21985-023</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:211px;">T- Cells isolation kit</td>
			<td style="width:157px;">Stemcell Technologies</td>
			<td style="width:135px;">19851</td>
			<td style="width:224px;">To isolate T cells</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:211px;">B- Cells isolation kit</td>
			<td style="width:157px;">Stemcell Technologies</td>
			<td style="width:135px;">19854</td>
			<td style="width:224px;">To isolate B cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Mouse T-Activator CD3/CD28 superparamagnetic beads</td>
			<td style="width:157px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">11452D</td>
			<td style="width:224px;">To activate T cells&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Cell isolation magnet</td>
			<td style="width:157px;">Stemcell Technologies</td>
			<td style="width:135px;">18000</td>
			<td style="width:224px;">To isolate T cells and remove the magnetic beads&#160;</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:211px;">AffiniPure F(ab&#39;)2 Fragment Goat Anti-Mouse IgG + IgM (H+L)</td>
			<td style="width:157px;">Jackson ImmunoResearch Laboratories, Inc.</td>
			<td style="width:135px;">115-006-068</td>
			<td style="width:224px;">To stimulate B cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Recombinant Murine IL-7</td>
			<td style="width:157px;">Peprotech</td>
			<td style="width:135px;">217-17&#160;</td>
			<td style="width:224px;">To support T-cell survival during activation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Recombinant CD40L</td>
			<td style="width:157px;">R&#38;D Systems</td>
			<td style="width:135px;">8230-CL/CF</td>
			<td style="width:224px;">To stimulate B cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Anti-mouse CD3 antibody</td>
			<td style="width:157px;">BD Pharmingen</td>
			<td style="width:135px;">561799</td>
			<td style="width:224px;">To stain T cells for flow-cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Anti-mouse CD19 antibody</td>
			<td style="width:157px;">BD Pharmingen</td>
			<td style="width:135px;">553786</td>
			<td style="width:224px;">To stain B cells for flow-cytometry</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:211px;">Biomed FXp</td>
			<td style="width:157px;">PerkinElmer Inc.</td>
			<td style="width:135px;">A31842</td>
			<td style="width:224px;">To re-suspend cells after 24 hours incubation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Opera Phenix High Content Screening System</td>
			<td style="width:157px;">PerkinElmer Inc.</td>
			<td style="width:135px;">HH14000000</td>
			<td style="width:224px;">To analyze GFP/Hoechst signal</td>
		</tr>
	</tbody>
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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.

Design of the HTS assay
Two important factors were taken into consideration when designing the herein fluorescent assay. First, we needed to replicate a physiological condition in which T- or B cell activation would represent an ailment (e.g. graft-versus-host disease). Second, the assessment of cellular activation should be performed using a sensitive and quantitative method. Fluorescence is nowadays one of the primary ch...

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

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 ...

The overall goal of this florescence-based lymphocyte assay is to identify new small molecule immunomodulators. This method can help answer key questions in the field of immunology about the mechanism of lymphocyte activation and inhibition. The main advantage of this phenotypic function and assay, that it can be adapted for drug discovery.Demonstrating the procedure will be Ahmed Fouda, a graduate student from our laboratory. Under a sterile work hood, disinfect the fur of a six to eight week old female Nurr77 GFP mouse with 70%ethanol, and place the animal on its right side. Next, make an incision in the skin and muscles of the upper left abdominal quadrant, and locate and remove the spleen, placing the tissue into splenocyte medium on ice.When all of the spleens have been collected, transfer the tissues to a 10 centimeter squared cell culture dish containing five milliliters of prewarmed splenocyte medium, and use a sterile syringe plunger to mash the tissues until the solution is turbid. Following extensive mashing in splenocyte medium, filter the cell suspension through a 70 micron cell strainer into a 50 milliliter tube to remove any debris, and centrifuge the resulting single cell suspension. Resuspend the pellet in two to three milliliters of red blood cell lysis buffer, sopping the reaction with five milliliters of RPMI, or another suitable buffer, after 20 to 30 seconds.Then collect the cells with another centrifugation, and resuspend the pellet in twp milliliters of splenocyte medium for cell counting. To isolate the T cells, centrifuge the cells again, and resuspend the pellet in serum free RPMI medium at a one times 10 to the seven cells per milliliter concentration. Transfer the cells to a five milliliter polystyrene tube, and set aside a 100 microliter aliquot of splenocytes to be used later for purity assessment.Next, add 50 microliters per milliliter of normal rat serum to the cells, followed by the addition of 50 microliters per milliliter of T cell isolation antibody cocktail with gentle mixing. After 10 minutes, incubate the cells with streptavidin conjugated magnetic beads for two and a half minutes. Then, add serum free medium to the cells for a final volume of two and a half milliliters, and place the tube in a cell isolation magnet for three minutes.At the end of the incubation, holding the tube in the magnet, pour the supernatant into a new polystyrene tube in one move. The T cells can then be counted. Seed T cells into a round bottom 96-well plate at a concentration of 2.5 times 10 to the fifth cells per well containing 200 microliters of fresh splenocyte medium per well.Add recombinant interleukin 7 and CD3/CD28 magnetic beads to the appropriate wells, and place the plate in a cell culture incubator. 12 hours later, gently triturate the cell suspension in each well to break up any bead cell aggregates. Then transfer the cell suspensions from each well into individual five milliliter polystyrene tubes.When all of the cells have been collected, place each tube of cells into the separation magnet for five minutes at a time, and decant the supernatants when the tube still inside the magnet, into new polystyrene tubes at the end up each incubation. Then collect the cells by centrifugation, and resuspend the pellets in fresh splenocyte medium at two times 10 to the sixth cells per milliliter concentrations. For small molecule high-throughput screening, plate 40 microliters of cells per well in a 384-well plate, and treat the appropriate wells with the drug or vehicle of choice for the desired treatment period in the cell culture incubator.30 minutes before the GFP analysis, stain the cells with the appropriate concentration of Hoechst solution, thoroughly distributing the stain with gentle pipetting. When all of the wells have been dyed, centrifuge the plates to collect the cells at the bottom of the wells, and allow the cells to rest for 15 minutes at room temperature. Load the plate according to the manufacturer's instructions.Then, set the objective at 40X or higher. Set camera number four for the UV lamp, and then set camera number one for laser 488. Load the plate to the high content screening system.Next, set the automated confocal high content screening system to read six to 10 fields per well with two sequential readings per field, at 488 nanometers and UV light. CD3 positive T cells represent approximately 30%of a given mouse spleen, demonstrating a 10%basal GFP expression. Following CD3/CD28 T cell receptor bead stimulation, a five to six fold increase in the percentage of GFP expression is observed.Likewise, CD19 positive B cells represent 50 to 55%of the total splenocytes with the basal GFP expression similar to that of non activated T cells. Interestingly, B cell receptor stimulation using anti-mouse IgG, IgM, and recombinant CD40L, triggers only a trivial increase in GFP expression. After magnetic bead isolation as just demonstrated, the isolated CD3 positive T cells still display a 10%basal GFP expression.CD3/CD28 bead stimulation however, induces a significant upregulation of the GFP response. Similarly, after B cell magnetic bead isolation, the purified activated B cells exhibit a higher expression of GFP compared to activated B cells in splenocytes. In this representative experiment, primary high-throughput screening of 4398 compounds revealed several activators and inhibitors of T cell activation.But the observed GFP expression of the stimulated T cells, 20 times higher than that measured for the unstimulated T cells. Allowing the generation of a window of action zone where the GFP inhibitory compounds could easily be detected. Once mastered, this technique can be completed in 36 to 48 hours if it is performed properly.While attempting this procedure, it is important to remember to count the cells after each step. Following this procedure, other methods like ELISA or western blotting can be performed to answer any other additional questions. This technique will pave the way for drug discovery immunomodulation in rodents.After watching this video, you should have a good understanding of how to isolate and activate T or B cells derived from the Nurr77 GFP mouse plates.

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