We are thankful for the funding sources from the Division of Biology and Johnson Cancer Research Center for BRIEF and IRA awards, respectively to GV. We also thank the K-INBRE postdoctoral award to RV. This work was supported in part by the Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103418. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health. We thank the anonymous reviewers for their helpful comments.

1. Setup and prerunning of standard liquid-phase IEF unit
<ol>
	<li>Assemble the liquid-phase IEF electrodes (anode-red button and cathode-black button) with their respective exchange membranes according to the instruction manual (see Table of Materials). Equilibrate the anion exchange membranes with 0.1 M NaOH and the cation exchange membranes with 0.1 M H3PO4 at least for 16 h when new membranes are used.
	<ol>
		<li>Store the membranes in electrolytes (0.1 M NaOH or 0.1 M H3PO4) between runs and do not allow to dry out.</li>
	</ol>
	</li>
	<li>Assemble the inner and outer portion of the electrode by aligning three oblong holes in the ion-exchange gaskets. Fill the electrodes with respective electrolytes (~25-30 mL) to prevent their membranes from drying out.
	<ol>
		<li>Do not add excess (more than 1/3rd of electrode chamber volume) electrolyte that may build up pressure inside the electrode and cause leakage.</li>
	</ol>
	</li>
	<li>Cover the sample collection ports with sealing tape that comes with the instrument assembly parts (see Table of Materials). The sample collection ports side can be identified by the two vertical metal aligning pins. Alternatively, use standard sealing tape to seal the ports.</li>
	<li>Assemble all parts of the focusing chamber assembly in sequence (anode electrode, nylon membrane core, focusing chamber and cathode electrode) over the ceramic cooling finger.</li>
	<li>Fill the focusing chamber with precooled distilled water (total volume of 60 mL for the standard IEF cell) using a 50 mL syringe.</li>
	<li>Connect the liquid-phase IEF instrument to a circulating cooling water at 4 &#186;C and prerun the unit at 15 W and 3,000 V for 3-5 min or until the voltage stabilizes. 
	NOTE: Generally, within one minute, the voltage will reach the maximum set values. Prerunning with distilled water helps to remove the residual ions from the focusing chamber and the nylon membrane core.</li>
	<li>Switch off the power source and remove the water from the cell using the fraction collector. Re-seal the collection ports with sealing tape. 
	NOTE: Now the instrument is ready for use in step 2.4.</li>
</ol>
2. Separation and purification of small molecules and peptides from Gymnema sylvestre&#160; extract
<ol>
	<li>Measure 0.6 g of plant extract and dissolve in distilled water (60 mL) by mixing in a roller tube for 5 min. 
	NOTE: Any biological sample that is soluble and free of salt can be used for separation and purification using this IEF instrument. Samples with buffering salt concentration up to 10 mM can be used with slightly decreased resolution. We are interested in the saponin family of triterpenoid compounds, the gymnemic acids, from G. sylvestre plant for their unique antifungal properties16.</li>
	<li>Centrifuge the solubilized plant extract at 10,000 x g for 5 min to remove insoluble particles.</li>
	<li>Transfer the supernatant (~60 mL) to an 80 mL centrifuge tube, and mix it with 0.6 mL of ampholyte (pH 3-10) to 1% (v/v).</li>
	<li>Follow steps 1.1 &#8211; 1.7 to prepare the liquid-phase IEF unit. The chamber is now ready to load the sample.</li>
	<li>Use a 50 mL syringe with a 1-1/2 inch 19 G blunt end needle (comes with the instrument) and load the prepared sample with ampholyte (60 mL total) in the cell through sample collection ports.</li>
	<li>Remove air bubbles from the sample cell by removing the focusing cell from the stand and tapping the electrode chamber to dislodge the bubbles. The presence of air bubbles will cause voltage and current fluctuations and affect the run.</li>
	<li>Connect the unit to the water coolant (4 &#176;C) and start fractionation with the power supply at a constant 15 W.</li>
	<li>Run the apparatus for 3 h or until the voltage reaches a constant value. 
	NOTE: As the sample starts focusing, the voltage will start climbing gradually until it reaches a constant value.</li>
	<li>After the run, prepare to collect the fractions in the harvest box (containing 20 plastic tubes, 12 mm x 75 mm, 5-6 mL volume) connected to the vacuum pump by pressing the harvest button ON in the IEF unit.</li>
	<li>Align the 20-collection pins with the 20-collection ports of the focusing cell that is sealed with the tape.</li>
	<li>Push the collection pins through the sealing tape and turn the vacuum pump ON simultaneously (see Figure 2B, 2C &#38; 2D). 
	NOTE: About 3 mL fractions from each chamber will be collected into the tubes for each time. Fractions can be used for subsequent downstream applications (SDS-PAGE for peptide quantification, TLC and bioassay for small molecules).</li>
</ol>
3. Separation and purification of proteins from&#160;&#160;C. albicans
<ol>
	<li>Grow single colony of C. albicans in yeast-peptone-dextrose (YPD) broth16 at 30 &#176;C with shaking (200 rpm) for overnight.</li>
	<li>Collect the yeast cells by centrifugation (10,000 x g for 5 min).</li>
	<li>Suspend C. albicans yeast cells in ammonium carbonate (1.89 g/L) buffer containing 1% (v/v) beta-mercaptoethanol (&#946;-ME) (1/10th of the culture volume) and rotate in a roller tube for 1 h at 5 &#176;C15.</li>
	<li>Remove the yeast cells by centrifugation (10,000 x g for 5 min) and filter the protein extract through a 0.45 &#181;m filter. 
	NOTE: Protein samples from C. albicans cytosol, membrane or cell wall can be prepared and used for IEF fractionation. Similarly, proteins from bacteria or other biological samples (animal tissue extract) can be used after removing any salt by appropriate methods (e.g., by dialysis or using desalting columns).</li>
	<li>Dialyze the protein extract in a dialysis tubing (3,500 MWCO) against water for 15 h at 4 &#176;C. Estimate the protein concentration by the Bradford dye-binding method using gamma globulin as a standard17.</li>
	<li>Use 500 mg of total protein in 60 mL of water containing 1% (v/v) ampholyte (pH 5-8) for the fractionation. 
	NOTE: Since a broad-range ampholyte (pH 3-10) does not enrich certain non-glucan attached cell wall proteins of C. albicans well, use a narrow-range (pH 5-8) ampholyte. An ampholyte concentration of up to 2% can be used if the sample protein concentration is more than 2 mg/mL; this minimizes protein aggregation during focusing. Always keep the samples, ampholytes, and water precooled on ice.</li>
	<li>Repeat steps 1.1- 1.7 to prepare the IEF unit and use the protein solution from step 3.6 to load into the IEF cell.</li>
	<li>After 4 h of focusing at a constant 15 W, harvest protein fractions (1-20) as described above (steps 2.9-2.11) and analyze on 12.5% SDS-PAGE after reducing and boiling the protein samples18.</li>
	<li>Stain the resolved proteins by staining the SDS-PAGE gel with the Coomassie blue dye (0.01%) solution for 2-3 h at room temperature on a rocker. Destain the gel and record the gel image using a gel imager. 
	NOTE: Coomassie blue dye (0.01%) can be prepared by mixing 0.01 g of Coomassie blue powder in a destaining solution containing 40% methanol and 10% acetic acid.</li>
</ol>
4. Bioactivity of purified small molecules from plant extract Gymnema sylvestre
<ol>
	<li>Grow C. albicans in yeast cells as in step 3.1.</li>
	<li>To prepare cell suspension, dilute overnight culture of C. albicans yeast cells (1/1000 dilution) into a fresh RPMI cell culture medium supplemented with 50 mM glucose. 
	NOTE: C. albicans converts from yeast growth to hyphae under hyphal inducing conditions (RPMI at 37 &#176;C). Gymnemic acid small molecules are shown to inhibit the conversion of yeast cells into hyphae under hypha inducing conditions16. We aimed to determine if liquid-phase-IEF-separated G. sylvestre extract contain these bioactive molecules.</li>
	<li>From the prepared cell suspension, add 90 &#956;L to each well of the 96-well plate.</li>
	<li>From each fraction (1-20 harvested fractions of G. sylvestre extract obtained from liquid-phase IEF, Figure 4), add 10 &#956;L into the wells with 90 &#956;L of the above cell suspension (step 4.2). Perform the assay in triplicate.</li>
	<li>Add 10 &#956;L of water that contains ampholyte (1%) into separate wells with 90 &#956;L of C. albicans yeast cell suspension as a negative control. 
	NOTE: Ampholytes have bioactivity potential and so it is essential to include an ampholyte control while performing any bioassay19.</li>
	<li>Incubate the 96-well plate at 37 &#176;C for 12 h and observe the inhibition of C. albicans yeast-to-hypha conversion under the microscope16. Also, determine the percentage of inhibition of yeast-to-hypha conversion.</li>
</ol>

<ol>
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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Method of Isoelectric Fractionation. , (1964).</li><li>Vesterberg, O., Svensson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoelectric+fractionation,+analysis,+and+characterization+of+ampholytes+in+natural+pH+gradients.+IV.+Further+studies+on+the+resolving+power+in+connection+with+separation+of+myoglobins.">Isoelectric fractionation, analysis, and characterization of ampholytes in natural pH gradients. IV. Further studies on the resolving power in connection with separation of myoglobins.</a> Acta Chemica Scandinavica. 20 (3), 820-834 (1966).</li><li>Righetti, P. 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The authors would like to declare no competing financial interests.

Small molecules from natural product sources (e.g., plants) include complex secondary metabolites that are highly diverse in chemical structure. They are believed to be involved in plant defense mechanisms. In addition, polypeptides are also present in plant tissues22. These natural product small molecules are rich sources of test molecules for drug discovery and development. However, the difficult and tedious methods required for their isolation and purification limit their use for therapeutic ap...

The purification of biomolecules from complex biological samples is an essential and often difficult step in biological experiments1. Isoelectric focusing (IEF) is well-suited for high resolution separation of complex biomolecules where carrier ampholytes travel according to their charge and establish the pH gradient in an electric field3. The first commercial carrier ampholyte for IEF was developed by Olof Vesterberg in 1964 and patented4,5. Carrier ampholytes are aliphatic oligo-amino oligo-carboxylic acid molecules of varying length and branching6. Subsequently, Vesterberg and others improved the carrier ampholytes for their expanded use in separating biomolecules6,7.
Methods to separate biomolecules include agarose and polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis (2-DE), isoelectric focusing, capillary electrophoresis, isotachophoresis and other chromatographic techniques (e.g., TLC, FPLC, HPLC)2. Liquid-phase IEF performed in an instrument called a &#8220;Rotofor&#8221; was invented by Milan Bier8. He pioneered the concept and design of this instrument and contributed extensively to the theory of electrophoretic migration. His team also developed a mathematical model of electrophoretic separation process for computer simulations9.
The liquid-phase IEF apparatus is a horizontally rotating cylindrical cell consisting of a nylon core divided into 20 porous compartments and a circulating water cooling ceramic rod. The porous chambers allow molecules to migrate through the aqueous phase between the electrodes and permit collection of purified samples under vacuum in fractions. This purification system can provide up to 1000-fold purification of a specific molecule in &#60;4 hours. A valuable feature of this instrument is that it can be applied as a first step for purification from a complex mixture or as a final step to achieve purity10. If the molecule of interest is a protein, another advantage is that its native conformation will be maintained during the separation.
The use of liquid-phase IEF has been reported widely for proteins, enzymes and antibody purification6,10,11,12,13,14. Here we describe the use of this approach for separating and purifying small molecules and peptides from the medicinal plant Gymnema sylvestre. This protocol will help researchers concentrate and purify active small molecules from a plant extract for downstream applications at low cost. In addition, we also demonstrate that enrichment of proteins from a complex protein extract from Candida albicans fungus15 in this IEF-based system as a second example.

<table>
	<tbody>
		<tr>
			<td>0.45 &#181;m syringe filter</td>
			<td>Fisher scientfic</td>
			<td>09-720-004</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>207861-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Lyte 3/10 Ampholyte</td>
			<td>Bio-Rad</td>
			<td>163-1113</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Lyte 5/8 Ampholyte</td>
			<td>Bio-Rad</td>
			<td>163-1192</td>
			<td></td>
		</tr>
		<tr>
			<td>Compact low temperature thermostat</td>
			<td>Lauda -Brinkmann</td>
			<td>RM 6T</td>
			<td>Set water cooling to 4 oC and it can be run even at 0 oC as when it passes through the Rotofor cooling core, the circulating water temperature will be around 5 or more depending on the voltage.</td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R</td>
			<td>Sigma-Aldrich</td>
			<td>B7920</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing (3,500 MWCO)</td>
			<td>Spectrum Spectra/Por</td>
			<td>132112T</td>
			<td></td>
		</tr>
		<tr>
			<td>Gymnema plant leaf extract powder (&#62;25% Gymnemic acids)</td>
			<td>Suan Farma, NJ USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Lab companion</td>
			<td>SI 300R</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>DM 6B</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini protean electrophoresis</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Mettler Toledo</td>
			<td>S20</td>
			<td>Useful to determine the pH of the Rotofor (liquid-phase IEF) fractions</td>
		</tr>
		<tr>
			<td>Rotofor</td>
			<td>Bio-Rad</td>
			<td>170-2972</td>
			<td>http://www.bio-rad.com/webroot/web/pdf/lsr/literature/M1702950E.pdf (Rotofor Instruction manual for assembling the unit)</td>
		</tr>
		<tr>
			<td>RPMI-1640 Medium</td>
			<td>HyClone</td>
			<td>DH30255.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sealing tape</td>
			<td>Bio-Rad</td>
			<td>170-2960</td>
			<td>Scotch tape may also be used.</td>
		</tr>
		<tr>
			<td>Sorvall legend micro 17 centrifuge</td>
			<td>Thermo scientific</td>
			<td>75002432</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP tissue culture plate -96 well flat bottom</td>
			<td>TPP</td>
			<td>TP92696</td>
			<td></td>
		</tr>
	</tbody>
</table>

separation of bioactive small molecules, peptides from natural sources and proteins from microbes by preparative isoelectric focusing (ief) method

Natural products derived from plants and microbes are a rich source of bioactive molecules. Prior to their use, the active molecules from complex extracts must be purified for downstream applications. There are various chromatographic methods available for this purpose yet not all labs can afford high performance methods and isolation from complex biological samples can be difficult. Here we demonstrate that preparative liquid-phase isoelectric focusing (IEF) can separate molecules, including small molecules and peptides from complex plant extracts, based on their isoelectric points (pI). We have used the method for complex biological sample fractionation and characterization. As a proof of concept, we fractionated a Gymnema sylvestre plant extract, isolating a family of terpenoid saponin small molecules and a peptide. We also demonstrated effective microbial protein separation using the Candida albicans fungus as a model system.

Separation and purification of small molecules and peptides from Gymnema sylvestre plant extract 
Using the preparative liquid-phase IEF method, we fractionated medicinal plant extracts and cell surface proteins from a human pathogenic fungus, C. albicans. A schematic of these fractionation protocols is shown in Figure 1.
From 20 fractions of G. sylvestre extract obtained from liquid-phase IEF, the dark-colored mole...

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Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing IEF Method

The objective is to fractionate and isolate bioactive small molecules, peptides from a complex plant extract, and proteins from pathogenic microbes by employing liquid-phase isoelectric focusing (IEF) method. Further, the separated molecules were identified and their bioactivity confirmed.

Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing (IEF) Method

Natural products derived from plants and microbes are a rich source of bioactive molecules. Prior to their use, the active molecules from complex extracts ...

separation-bioactive-small-molecules-peptides-from-natural-sources

Research

JoVE Journal

Immunology and Infection

9.3K Views.  Kansas State University. The objective is to fractionate and isolate bioactive small molecules, peptides from a complex plant extract, and proteins from pathogenic microbes by employing liquid-phase isoelectric focusing (IEF) method. Further, the separated molecules were identified and their bioactivity confirmed.

Video: Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing IEF Method

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the &asymp;40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. 
When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte.
Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite.
Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing.

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

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

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

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

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

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

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

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 ...