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1. Preparation of Splenocyte Medium and Flow-cytometry Buffer
<ol>
	<li>Perform all steps under a 70% ethanol-cleaned biological hood.</li>
	<li>Remove 70 mL of pre-warmed Roswell Park Memorial Institute (RPMI) 1640 1x medium (commercially available and supplied in filtered 500 ml volume sterile bottles) and place in a sterile tube. The removed medium will be used later in the protocol.</li>
	<li>Supplement the remaining 430 mL of RPMI 1640 1x with 50 ml inactivated fetal bovine serum (FBS), 5 mL penicillin/streptomycin, 5 mL N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), 5 ml non-essential amino acids, 5 mL sodium pyruvate, and 0.05 mL filtered (1M) 2-mercaptoethanol. 
	NOTE: Pre-warm splenocyte medium using a water bath set at 37 &#186;C to avoid heat shocking cells. This is important to avoid induction of cellular apoptosis.</li>
	<li>To prepare the flow-cytometry buffer, add 2 mL of FBS to 98 ml phosphate buffered saline (PBS) and keep refrigerated until use (preferably within three days).</li>
</ol>
2. Generation of Splenocyte Cell Suspension from&#160;Nur77GFP&#160;Mouse Spleens
<ol>
	<li>Aseptically isolate the spleen from a 6-8 week old female Nur77GFP&#160;mouse.
	<ol>
		<li>To achieve this, work under a sterile hood. Soak the sacrificed mouse fur, scissors, and forceps in 70% ethanol.</li>
		<li>Lay the mouse on its right side, and cut the skin and muscles in the upper left abdominal quadrant. Inspect the incision area to visibly locate the spleen then cut it out. Keep the spleen in splenocyte medium on ice until ready to perform the next step.</li>
	</ol>
	</li>
	<li>Place the spleen(s) in a 10 cm2&#160;cell culture petri dish containing 5 ml of pre-warmed splenocyte medium.</li>
	<li>Mash the spleen using a sterile syringe plunger until the solution is turbid. A spleen collagen matrix should remain by the end of the procedure.</li>
	<li>Following extensive mashing in splenocyte medium, collect the cell suspension and pass it through a 70 &#181;m cell strainer placed on a 50 mL tube to filter out any debris or clots.</li>
	<li>Centrifuge at 500 x g for 5 min. After discarding the supernatant, re-suspend the cell pellet in 2-3 mL of in-house produced or commercial red blood cell lysis buffer. Following pipetting (2-3 times) let the suspension rest for 20-30 s.</li>
	<li>Add 5 mL of PBS or a suitable buffer of choice then centrifuge the cell suspension for 5 min at 500 x g.</li>
	<li>Remove the supernatant (should be red in color as it contains lysed red blood cells) and re-suspend the cell pellet in 2 ml of splenocyte medium.</li>
	<li>Using a hemocytometer, count the number of cells stained with trypan blue to differentiate between live and dead (necrotic) cells.</li>
</ol>
3. T-cell Isolation from the Splenocyte Cell Suspension
<ol>
	<li>Centrifuge the cell suspension for 5 min at 500 x g. Resuspend the cells in serum-free RPMI medium (50 ml from step 1.3) to obtain a cellular concentration of 10 x 106&#160;cells/mL.</li>
	<li>Transfer the cells to a 5 mL polystyrene tube and set aside a 100 &#181;L aliquot of splenocytes for purity assessment/comparison at the end of the purification step.</li>
	<li>Add normal rat serum to the cell suspension at 50 &#181;L/mL followed by a T cell isolation antibody cocktail (50 &#181;L/mL).</li>
	<li>Mix the cell suspension and let it rest for 10 min. This step allows the antibody cocktail to bind all unwanted cells.</li>
	<li>Add the streptavidin rapid spheres (magnetic beads) for 2.5 min, then bring the volume to 2.5 mL using the serum-free medium.</li>
	<li>Place the tube in a cell isolation magnet for 3 min.</li>
	<li>Transfer the T cell suspension into a new polystyrene tube by holding the magnet and pouring out the solution in one move. 
	NOTE: The isolated suspension contains the purified T cells. All unwanted cells are held on the side of the tube bound to the magnetic streptavidin beads.</li>
	<li>Count the isolated T cells using trypan blue and a hemocytometer. 
	NOTE: At this step, the purity of the isolated T cells can be verified (optional) by analyzing the percentage of CD3+&#160;events before and after T cell isolation by flow cytometry.
	<ol>
		<li>Briefly, centrifuge 1 ml of splenocyte cell suspension at 500 x g. Discard the supernatant and suspend the cells in a flow cytometry buffer at a concentration of 1 x 106&#160;cells/mL.</li>
		<li>Add fluorescent-tagged anti-mouse CD3 antibody at a concentration of 1:100. Incubate at 4 &#176;C for 30 min. Wash once, centrifuge, and re-suspend in flow cytometry buffer (400 &#181;L) for analysis by flow cytometry.</li>
	</ol>
	</li>
</ol>
4. T-cell Activation and the Induction of green fluorescent protein (GFP) Expression
<ol>
	<li>Seed the T cells, in suspension, into a round-bottomed 96-well plate at a concentration of 2.5 x 105&#160;cells/well.</li>
	<li>Add recombinant interleukin (IL)-7 at 2 ng/mL and CD3/CD28 magnetic beads (25 &#181;L/106&#160;cells). Keep a portion of the T cells untreated with beads to serve as a negative control for later measurements that represent non-activated T cells.</li>
	<li>Twelve hours later, harvest the T cells from the 96-well plate by gently pipetting the cell suspension in each well up and down to break any bead-cell complex/aggregates. Collect the suspension from all wells into a 5 mL polystyrene tube.</li>
	<li>Place the tube containing the suspension inside the same cell isolation magnet used previously for the purification of T cells and allow it to rest for 5 min.</li>
	<li>Transfer the T cell suspension into a new polystyrene tube by holding the magnet and pouring out the solution in one move.</li>
	<li>Centrifuge the cells at 500 x g for 5 min. Re-suspend the cells in fresh splenocyte medium to get a concentration of 2 x 106&#160;cells/mL.</li>
	<li>Assess GFP expression intensity by flow-cytometry (optional for quality control) at 12 to 24 h post-stimulation in comparison with non-activated T cells.12 
	NOTE: The GFP fluorescence is intrinsic to the Nur77GFP&#160;T cells and is manifested upon successful activation of the T-cell receptor, TCR.</li>
	<li>For assessment of viability and activation (optional) by microscopy, stain the activated cells with Hoechst 30 min prior to analysis at a concentration of 0.2 &#181;g/ml. Add an adequate volume of the cell suspension to a cover slide or a well of a flat-bottomed black-sided 384-well plate and examine under a fluorescence microscope to assess living cells. Dead cells will not retain nuclear staining.</li>
</ol>
5. High Throughput Screening (HTS) of Small Molecules
<ol>
	<li>Prepare a cell suspension at 2 x 106&#160;cells/mL of the activated T cells (magnetic beads or antibodies/CD40L) or non-activated groups.</li>
	<li>Plate 75,000 cells/well in a 384-well plate (volume of 40 &#181;L). Use a flat-bottomed black-sided 384-well plate or a microscope cover slide for viability and activation assessment by fluorescent microscopy (optional quality control step prior to HTS) using Hoechst stain (refer to steps 4.8 for details).</li>
	<li>Manually or using an automated system, add the drugs of choice (dissolved in 0.5% dimethyl sulfoxide, DMSO) to each well.</li>
	<li>Add the vehicle (DMSO) to positive (activated) and negative (non-activated) control wells. Adjust DMSO concentration to a maximum of 0.5%.</li>
	<li>Incubate the plates for 24 h (or incubation time of choice) at 37 &#186;C and 5% carbon dioxide, CO2.</li>
	<li>On the day of the screening, dilute the Hoechst 33342 stain solution (1:3,333; for&#160;e.g. add 10 &#181;L to the cells in the 384-well plates to generate a total volume of 50 &#181;L). Stain 30 min before GFP analysis by adding Hoechst solution to achieve a concentration of 0.2 &#181;g/mL.</li>
	<li>Gently pipette the cells up and down to obtain a homogenous distribution in each well. 
	NOTE: This step is important in the case of dispensing the drugs (step 5.3) using an automated system as the cells tend to accumulate at one side of the well, opposite to the direction of the flow.</li>
	<li>Spin the plates at 45 x g for 3 min at room temperature.</li>
	<li>Leave the plates to rest for 15 min at room temperature.</li>
	<li>Perform plate(s) read-outs, using the automated confocal high content screening (HCS) system.&#160;Load the plate to the machine. Set the objective at 40X or higher magnification. Use camera #4 for Hoechst (UV lamp) and camera #1 for GFP (laser 488). Set up the machine to read 6-10 fields per well. Adjust the machine to do two sequential readings per field at 488 nm (to read GFP) and UV light (to read Hoechst). Set the objective at 40X or higher magnification. 
	NOTE: The system is a computerized microscope that does not require any adjustments. The focal distance, the intensity of the incident light, and the time of exposure are all set up automatically by the machine.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Nur77GFP mice</td>
			<td>The Jackson Laboratory</td>
			<td>Mouse strain No. 016617</td>
			<td>An in house colony was established at our animal facility&#160;</td>
		</tr>
		<tr>
			<td>96 wells-U culture plates, sterile</td>
			<td>VWR International</td>
			<td>10062-902</td>
			<td>T-cell activation using the magnetic beads</td>
		</tr>
		<tr>
			<td>70&#181;m cell strainer, sterile</td>
			<td>Corning Inc.</td>
			<td>352350</td>
			<td>Generation of splenocytes cell suspension</td>
		</tr>
		<tr>
			<td>5 ml polystyrene round bottom tubes, sterile</td>
			<td>Corning Inc.</td>
			<td>352058</td>
			<td>Generation of splenocytes cell suspension</td>
		</tr>
		<tr>
			<td>50 ml polypropylene conical bottom tubes, sterile</td>
			<td>VWR International</td>
			<td>89039-656&#160;</td>
			<td>Generation of splenocytes cell suspension</td>
		</tr>
		<tr>
			<td>10 ml syringe without needle, sterile</td>
			<td>Becton, Dickinson and Company</td>
			<td>305482</td>
			<td>To mash the spleen</td>
		</tr>
		<tr>
			<td>5 ml cell culture dish, sterile</td>
			<td>Greiner Bio-One</td>
			<td>627 160</td>
			<td>To mash the spleen</td>
		</tr>
		<tr>
			<td>Penicillin- Streptomycin (10,000 U/mL)</td>
			<td>WISENT Inc.</td>
			<td>450-200-EL&#160;&#160;</td>
			<td>Component of the splenocyte media</td>
		</tr>
		<tr>
			<td>RPMI 1600 with sodium bicarbonate and L- glutamine</td>
			<td>WISENT Inc.</td>
			<td>350-002-CL&#160;</td>
			<td>Component of the splenocyte media</td>
		</tr>
		<tr>
			<td>MEM non-essential amino acids</td>
			<td>WISENT Inc.</td>
			<td>321-010-EL&#160;</td>
			<td>Component of the splenocyte media</td>
		</tr>
		<tr>
			<td>HEPES free acid 1 M</td>
			<td>WISENT Inc.</td>
			<td>330-050-EL&#160;</td>
			<td>Component of the splenocyte media</td>
		</tr>
		<tr>
			<td>Sodium pyruvate solution (100mM)</td>
			<td>WISENT Inc.</td>
			<td>600-110-EL&#160;</td>
			<td>Component of the splenocyte media</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)&#160;</td>
			<td>WISENT Inc.</td>
			<td>080-910&#160;</td>
			<td>Component of the splenocyte media and flow-cytometry buffer</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>WISENT Inc.</td>
			<td>311-010-CL</td>
			<td>Component of flow-cytometry buffer</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol (55mM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>21985-023</td>
			<td>Component of the splenocyte media</td>
		</tr>
		<tr>
			<td>T- Cells isolation kit</td>
			<td>Stemcell Technologies</td>
			<td>19851</td>
			<td>To isolate T cells</td>
		</tr>
		<tr>
			<td>Mouse T-Activator CD3/CD28 superparamagnetic beads</td>
			<td>Thermo Fisher Scientific</td>
			<td>11452D</td>
			<td>To activate T cells&#160;</td>
		</tr>
		<tr>
			<td>Cell isolation magnet</td>
			<td>Stemcell Technologies</td>
			<td>18000</td>
			<td>To isolate T cells and remove the magnetic beads&#160;</td>
		</tr>
		<tr>
			<td>Recombinant Murine IL-7</td>
			<td>Peprotech</td>
			<td>217-17&#160;</td>
			<td>To support T-cell survival during activation</td>
		</tr>
		<tr>
			<td>Anti-mouse CD3 antibody</td>
			<td>BD Pharmingen</td>
			<td>561799</td>
			<td>To stain T cells for flow-cytometry</td>
		</tr>
		<tr>
			<td>Biomed FXp</td>
			<td>PerkinElmer Inc.</td>
			<td>A31842</td>
			<td>To re-suspend cells after 24 hours incubation</td>
		</tr>
		<tr>
			<td>Opera Phenix High Content Screening System</td>
			<td>PerkinElmer Inc.</td>
			<td>HH14000000</td>
			<td>To analyze GFP/Hoechst signal</td>
		</tr>
	</tbody>
</table>

a fluorescence-based assay using engineered lymphocytes for screening immunomodulatory compounds

Source: Fouda, A., et al., <a href="https://app.jove.com/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.

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

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 ...

Begin with a fluorescent-compatible multi-well plate containing a suspension of transgenic mouse-derived T lymphocytes.
These lymphocytes contain a green fluorescent protein or GFP expression cassette, controlled by T cell receptor or TCR engagement with immunomodulatory compounds.
Treat each well with a solution containing immunomodulatory compounds with different potentials.
During incubation, immunomodulatory compounds interact with TCRs on T lymphocytes.
TCR interaction with a stimulating compound triggers the activation of the gene cassette, elevating GFP expression.
In contrast, TCR interaction with an inhibitory compound inhibits the gene cassette and reduces the GFP expression.
Next, overlay the cells with nucleic acid fluorescent dye to stain the nuclei.
Centrifuge the plate to settle the cells for accurate fluorescence measurement.
Under a fluorescence microscope, viable T lymphocytes exhibit two fluorescence signals corresponding to cell nuclei and green fluorescent proteins.
The intensified green fluorescence signifies successful stimulation of T lymphocytes, while a faint intracellular green fluorescence signal confirms the inhibitory effect of immunomodulatory compounds on T lymphocytes.

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Universidad San Sebastian

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Specialized Assays

72 Views. Fonte: Fouda, A., et al., Un test dei linfociti basato sulla fluorescenza adatto per lo screening ad alto rendimento di piccole molecole. J. Vis. Exp., (2017). Questo video dimostra lo screening di composti immunomodulatori tramite test basati sulla fluorescenza. I linfociti T transgenici derivati da topi con cassette di espressione proteica fluorescente verde sono trattati con questi composti. I segnali GFP potenziati e ridotti consentono l'identificazione degli effetti stimolatori e inibitori...

Video: Un test basato sulla fluorescenza che utilizza linfociti ingegnerizzati per lo screening di composti immunomodulatori - Concept

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median survival following diagnosis is less than 15 months with maximal surgical resection, radiation, and temozolomide chemotherapy. The challenges inherent in developing more effective GBM treatments have become increasingly clear, and include its unyielding invasiveness, its resistance to standard treatments, its genetic complexity and molecular adaptability, and subpopulations of GBM cells with phenotypic similarities to normal stem cells, herein referred to as glioblastoma stem cells (GSCs). Because GSCs are required for tumor growth and progression, differentiation-based therapy represents a viable treatment modality for these incurable neoplasms. 
The following protocol describes a collection of procedures to establish a high throughput screening platform aimed at the identification of small molecules that promote GSC astroglial differentiation. At the core of the system is a glial fibrillary acidic protein (GFAP) differentiation reporter-construct. The protocol contains the following general procedures: (1) establishing GSC differentiation reporter lines; (2) testing/validating the relevance of the reporter to GSC self-renewal/clonogenic capacity; and (3) high-capacity flow-cytometry based drug screening. 
The screening platform provides a straightforward and inexpensive approach to identify small molecules that promote GSCs differentiation. Furthermore, utilization of libraries of FDA-approved drugs holds the potential for the identification of agents that can be repurposed more rapidly. Also, therapies that promote cancer stem cell differentiation are expected to work synergistically with current "standard of care" therapies that have been shown to target and eliminate primarily more differentiated cancer cells.

An efficient screening protocol is presented for the identification of small molecules that promote astroglial differentiation in glioblastoma stem cells (GSCs). The assay is based on a stem cell differentiation reporter whereby the expression of the enhanced GFP (eGFP) is driven by the human GFAP promoter.

Farmaco a base di citometria a flusso sistema di Screening per l'identificazione di piccole molecole che promuovono la differenziazione cellulare delle cellule staminali di Glioblastoma

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

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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.

Creazione di una configurazione high-throughput screening per piccole molecole che modulano c-di-GMP Segnalazione di<em&gt; Pseudomonas aeruginosa</em

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

Immunologia e infezioni

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Screening ad alto rendimento per i modulatori chimici di Genes post-trascrizionale regolamentati

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

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

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