This study was supported by Max McGee National Research Center for Juvenile Diabetesat Medical College of Wisconsin and Children's Research Institute of Wisconsin. The funders had no role in study design, data collection and analysis, or preparation of the manuscript.

1. Before setting up a suppression assay, one needs to coat tosylactivated beads with anti-human CD3 (clone UCHT1, final concentration 1&mu;g/ml) for cell stimulation and afterwards check whether the beads are efficiently coated by setting up an in vitro proliferation assay using human T cells
<ol>
<li>Take 1ml of M-450 tosylactivated beads from original vial, place in magnetic stand and hold until all the beads have adhered to the side of the tube. Remove the buffer while tube is still in the magnetic stand. Take tube out of magnetic stand and add 1ml of buffer1, place the tube in magnetic stand again and remove the buffer1 while tube is in the magnetic stand; resuspend beads in 1ml of buffer1 and add 40&mu;l of anti-human CD3, agitate at 37&deg;C for 15 minutes, add 0.1%w/v BSA and continue agitating for next 16 hours.</li>
<li>Wash the beads in buffer2 twice for 5 minutes at 2-8&deg;C and once in buffer3 for 5 minutes at 2-8&deg;C using magnetic stand as explained above; remove the buffer and resuspend the beads in 1ml of buffer2; the beads are 4x108/ml final concentration and ready for use.</li>
<li>Aliquot 50,000 and 25,000 PBMC/well in triplicates in a 96-well plate and add variable number of CD3-coated beads (4x108beads/ml, CD3 1&mu;g/ml, for example 1, 2, 3, 4, 5 beads/cell) in order to determine the optimal number of beads per cell. After 72 hours in culture, add 1&mu;Ci [3H] thymidine and continue incubating at 37&deg;C for the next 16 hours. Harvest cells in Multiscreen harvest plate (Millipore), add scintillation liquid and read counts per minutes (cpm)/well using Top Count NXT (Packard, CT). Use the beads/cell ratio when cpms are above 5000 but less than 15000 to avoid overstimulation of Tregs, which might lose suppressive function. Usually, ratio of 3 beads/cell stimulates both responder and Treg cells in all subject groups tested so far1-4.</li>
</ol>
2. PBMC isolation from whole blood from healthy donors or from human leukopacks or buffy coat (BC) usually taken from healthy volunteers and available free of charge from local Blood Transfusion Centers (Figure 1)
<ol>
<li>Dilute the BC (&tilde;50ml) 1:6 with PBS (add 250ml). Now there is 300ml total volume. Slowly layer 25ml of diluted BC on top of 15ml Ficoll-Paque PLUS added to 50ml Falcon tubes, without disturbing the layers. Centrifuge at 800xg (1400rpm in a Sorvall centrifuge with swinging bucket rotor SH-3000) for 30 minutes at 4&deg;C, with brakes turned off. </li>
<li>Carefully collect the PBMC layer (intermediate phase) and transfer it to a fresh 50ml Falcon tubes. Wash PBMC by filling the tubes up to 50ml with DPBS. Collect 2 cell pellets into one tube. Centrifuge at 400xg for 10 min at 4&deg;C. Repeat washing step twice, each time combining 2 cell pellets into a single tube. Combine all cell pellets into a single 50ml tube. </li>
<li>Count PBMC using Trypan Blue exclusion test. Make a 1:10 dilution in Trypan Blue by adding 20&mu;l of PBMC to 180&mu;l of Trypan Blue stain. Mix well and take 20&mu;l to count in hemacytometer immediately. Count numbers of all unstained and only blue stained cells located in two square blocks each containing 16 smaller squares. Take the average of both numbers and multiply by 10. Divide this number by 100 to get the number of PBMC/ml. Percentage of viable cells calculate as [1-(number of blue cells/number of total cells)x100]. Proceed if viability is &ge;95%.</li>
</ol>
3. MACS pre-sort of CD4 T cells
<ol>
<li>Before proceeding, transfer 1ml of PBMC into a new tube, add 4ml of media to prepare cells for irradiation with 5000rad- these will be antigen-presenting cells (APC). </li>
<li>Centrifuge the rest of the cells at 250xg in PBS/2mM EDTA/0.5%BSA buffer for 10min at 4&deg;C. Pour off supernatant, and resuspend cells in 4ml of PBS/2mM EDTA/0.5%BSA buffer.</li>
<li>Add 200&mu;l of MACS anti-CD4 microbeads and incubate at 4&deg;C for 20 minutes.</li>
<li>Wash by adding 40ml of PBS/2mM EDTA/0.5%BSA buffer and centrifuge at 250xg for 10min at 4&deg;C. Pour off supernatant and resuspend in 8ml of degassed, room temperature PBS/2mM EDTA/0.5%BSA buffer.</li>
<li>Filter-separate the cell suspension using Pre-separation filters before loading them on an LS column.</li>
<li>Split the cell suspension and layer 4ml each over calibrated LS column (with 3ml of deggassed PBS/2mM EDTA/0.5%BSA buffer). LS column is fixed in the MidiMACS separator. Before cell suspension runs out, either re-run flowthrough or add 3ml of deggassed room temperature PBS/2mM EDTA/0.5%BSA buffer and let the buffer run through. Add more buffer until it comes out clear.</li>
<li>Pipette 5ml deggassed buffer onto the LS column, remove LS Column from the MidiMACS separator and place into a new sterile 15ml collection tube letting &tilde;1.0ml run through. Put the plunger into the column and slowly push the rest of the volume out.</li>
<li>Do the same with both LS columns and combine the two CD4+ fractions. Add up to 50ml PBS and count cells. Expected yield is up to 5x108 cells. Centrifuge at 400xg for 10 minutes and resuspend cells well in 2ml PBS/2mM EDTA/0.5%BSA buffer.</li>
</ol>
4. Fluorescent Activated Cell Sorting (FACS) isolation (Figure 2)
<ol>
<li>Make a cocktail of antibodies to CD markers to the following cell surface markers (keep protected from light): 20 &mu;l of anti-human CD8-FITC (clone RPA-T8), 20 &mu;l of anti-human CD14-FITC (clone M5E2; LPS receptor), 20 &mu;l of anti-human CD32-FITC (clone FLI8.26; Fc&gamma;R-type II) and 6 &mu;l of anti-human CD116-FITC (clone M5D12; GM-CSFR&alpha; chain) and, alternatively, add 40&mu;l anti-human CD4-APCCy7 (clone RPA-T4).</li>
<li>Take 5&mu;l out of the 2ml cell suspension and stain with 2&mu;l of stain cocktail; this is a tube for determining the threshold (Fluorochrome Minus One-FMO)5. </li>
<li>Add 50 &mu;l of anti-human CD25-PE (clone M-A251; IL-2R&alpha;) to the stain cocktail, and add the cocktail to cell suspension and incubate at 4&deg;C 30 minutes. Wash cells in PBS buffer, centrifuge at 400xg for 10 minutes and resuspend cells to a cell concentration of 107/ml. </li>
<li>Prepare unstained cells and cells or beads stained with single fluorochrome to use as compensation control for cell sorting on FACS Aria (BD Biosciences, San Jose, NJ).</li>
<li>Acquire cells in the FMO tube which will allow the user to set the threshold for sorting of CD25+ T cells (Figure 2a). Set a gate around FITC-negative cells to exclude FITC-positive cells comprising monocytes, macrophages and all other CD4+-non-T cells (Figure 2b).</li>
<li>In a separate plot, draw gates for CD25-, CD25low and CD25high T cells-Tregs (top 1% of cells expressing the highest number of CD25) (Figure 2c). Cell subsets typically show high purity (Figure 2d, 2e and 2f).</li>
<li>Centrifuge collection tubes with cells at 400xg for 10 minutes and keep them on ice until plating.</li>
</ol>
5. Set up cell culture in 96-well plate (scheme attached as Table1) in 200&mu;l/well
<ol>
<li>Aliquot 50&mu;l of CD3-coated beads (1 &mu;g/ml) calculated to be 3 beads/responder cell in a well, resuspended in complete media with 10% pooled human AB serum in U-bottom 96-well plates. (For example, to make 2ml media with CD3-coated, take 7.5&mu;l from stock of CD3-coated beads -final concentration 4x108/ml and dilute in 2ml of media; every 50&mu;l will contain 75,000 beads-3beads/cell).</li>
<li>Dilute irradiated APC to 5x105/ml cell concentration and add 50&mu;l (will contain 2.5 x 104 cells) into each well with previously added stimulation, including wells labeled as "Tregs only", "APC only" and "media only" in Table 1.</li>
<li>Add 2.5 x 104/well CD4CD25- or CD4CD25low T cells in triplicates following design in Table 1.</li>
<li>Add Tregs to co-cultures (row B, Table 1) in the ratio 1:10 (2,500 Treg cells) and to wells labeled as "Tregs only" and incubate the plate at 37&deg;C in CO2 incubator with 5% CO2, in saturated humidity for 72 hours.</li>
<li>Pulse wells with 1&mu;Ci [3H] thymidine and continue incubation at 37&deg;C for next 16 hours.</li>
</ol>
6. Harvesting and counting 
<ol>
<li> Harvest cells on multiscreen harvest plate using Packard filtermate harvester or alternative system.</li>
<li>Add scintillation liquid (Microscint 20), cover harvesting plate with transparent plastic cover in preparation for the final step.</li>
<li>Read counts per minutes (cpm)/well using Top Count NXT (Packard, CT) or alternative system.</li> 
</ol>
7. Computing percentage of suppression
<ol>
<li>As cells were cultured in triplicates, average is calculated for each condition. If the coefficient of variation is &gt;30%, the outlier is eliminated and only cpm from two wells are averaged. Percentage of suppression is obtained by computing [(s-c)/s] x 100%, where s=cpm in single culture and c=cpm in co-culture.</li>
<li>As na&iuml;ve (CD25-) and in vivo activated (CD25low) effector T cells are plated as responder T cells, the difference in the ability of Tregs to suppress each of these subsets is captured and used as a potential functional prognostic indicator based on the premise that activated cells are harder to suppress.</li>
</ol>
8. Representative Results: 
Great variability in the methods used and results derived from in vitro human suppression assay prompted us to perform a comprehensive study of conditions influencing the assay1. We have developed an assay that tests not only Treg function, but also their purity, considering the low ratio between Tregs:Teffs (1:10), which we determined earlier6. In addition, Tregs differ in their ability to successfully suppress na&iuml;ve and in vivo-activated T cells even in healthy subjects, as shown in Figure 3 and in our previous studies2,4, which becomes more prominent if immune balance is compromised, as in subjects at risk to develop T1D. The assay worked very well in the study where we compared suppressive function of natural (nTregs), inducible (iTregs) and in vitro expanded nTregs, allowing us to compare their function between healthy control, recent-onset (RO) T1D and longstanding (LS) T1D subjects. We concluded that RO T1D subjects had better capacity of generating functional both iTregs and expanded nTregs compared to LS T1D and healthy control subjects7. Thus, this assay can be used as an excellent tool in the recognition of both an early and late state of immune imbalance. 
Scheme 1 Schematic presentation of the steps involved in in vitro suppression assay
<img src="/files/ftp_upload/3071/3071fig1.jpg" alt="Figure 1"/> Figure 1. Steps of the in vitro suppression assay presented with photographs 
<img src="/files/ftp_upload/3071/3071fig2.jpg" alt="Figure 2"/> Figure 2. Gating strategy in FACS cell isolation. a) CD25+ threshold was adjusted according to Fluorochrome Minus One (FMO), b) cells were gated as FITC-negative, c) FITC-negative cells were further gated and collected as CD+CD25-, CD+CD25low and CD+CD25high (Tregs) shown with percentages, d) FACS sorted Tregs after sorting, e) FACS sorted CD+CD25low after sorting, and f) FACS sorted CD4+CD25- T cells after sorting
<img src="/files/ftp_upload/3071/3071fig3.jpg" alt="Figure 3"/> Figure 3. Representative results of healthy subjects a) Representative results of counts per minute (cpm) of healthy subjects presented as single cultures for all cell subsets involved (na&iuml;ve-CD25-, in vivo activated-CD25low, antigen-presenting cells-APC and regulatory T cells-Treg) as well as co-cultures of responder T cells (CD25- or CD25low) and Tregs. b) Percentage of suppression of each CD25- and CD25low responder T cells by autologous Tregs is presented for healthy control subjects (n=4). Suppression was computed as [(s-c)/s] x 100%, where s=cpm in single culture and c=cpm in co-culture. Although slight difference in capacity of Tregs to suppress responder T cells was noticed, it was not significant (paired t-test p=0.08). c) Presented are cpm of at risk subjects for each single culture, including CD25- and CD25low as responder T cells as well as APC and Tregs, and co-cultures where each responder T cell subset is seeded with Tregs (CD25-/Tregs and CD25low/Tregs). d) Percentage of suppression of each CD25- and CD25low responder T cells by autologous Tregs is presented for at risk subjects (n=4). The difference in capacity of Tregs to suppress CD25- versus CD25low responder T cells was significant (paired t-test p=0.04). 
Table 1. Schematic set up of in vitro suppression assay
<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>1-3</td>
			<td>4-6</td>
			<td>7-9</td>
			<td>10-12</td>
		</tr>
		<tr>
			<td>A</td>
			<td>CD4CD25-</td>
			<td>CD4CD25low</td>
			<td>media only</td>
			<td>media only</td>
		</tr>
		<tr>
			<td>B</td>
			<td>CD4CD25- /Tregs</td>
			<td>CD4CD25low /Tregs</td>
			<td>Tregs only</td>
			<td>APC only</td>
		</tr>
		<tr>
			<td>C</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>D</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>E</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>F</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>G</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>H</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

<ol>
	<li>Barge, A., Cravotto, G., Gianolio, E., Fedeli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+determine+free+Gd+and+free+ligand+in+solution+of+Gd+chelates.+A+technical+note.">How to determine free Gd and free ligand in solution of Gd chelates. A technical note.</a> Contrast Med. Mol. Imaging. 1, 184-188 (2006).</li><li>Nagaraja, T. N., Croxen, R. L., Panda, S., Knight, R. A., Keenan, K. A., Brown, S. L., Fenstermacher, J. D., Ewing, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+arsenazo+III+in+the+preparation+and+characterization+of+an+albumin-linked,+gadolinium-based+macromolecular+magnetic+resonance+contrast+agent.">Application of arsenazo III in the preparation and characterization of an albumin-linked, gadolinium-based macromolecular magnetic resonance contrast agent.</a> J. Neurosci. Methods. 157, 238-245 (2006).</li><li>Supkowski, R. M., Horrocks, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+determination+of+the+number+of+water+molecules,+q,+coordinated+to+europium(III)+ions+in+solution+from+luminescence+decay+lifetimes.">On the determination of the number of water molecules, q, coordinated to europium(III) ions in solution from luminescence decay lifetimes.</a> Inorg. Chim. Acta. 340, 44-48 (2002).</li><li>Menjoge, A. R., Kannan, R. M., Tomalia, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrimer-based+drug+and+imaging+conjugates:+design+considerations+for+nanomedical+applications.">Dendrimer-based drug and imaging conjugates: design considerations for nanomedical applications.</a> Drug Discovery Today. 15, 171-185 (2010).</li><li>Que, E. L., Chang, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responsive+magnetic+resonance+imaging+contrast+agents+as+chemical+sensors+for+metals+in+biology+and+medicine.">Responsive magnetic resonance imaging contrast agents as chemical sensors for metals in biology and medicine.</a> Chem. Soc. Rev. 39, 51-60 (2010).</li><li>Uppal, R., Caravan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+probes+for+cardiovascular+MR+imaging.">Targeted probes for cardiovascular MR imaging.</a> Future Med. Chem. 2, 451-470 (2010).</li><li>Major, J. L., Meade, T. J. B. i. o. r. e. s. p. o. n. s. i. v. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioresponsive,+cell-penetrating,+and+multimeric+MR+contrast+agents.">Bioresponsive, cell-penetrating, and multimeric MR contrast agents.</a> Acc. Chem. Res. 42, 893-903 (2009).</li><li>Datta, A., Raymond, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gd-hydroxypyridinone+(HOPO)-based+high-relaxivity+magnetic+resonance+imaging+(MRI)+contrast+agents.">Gd-hydroxypyridinone (HOPO)-based high-relaxivity magnetic resonance imaging (MRI) contrast agents.</a> Acc. Chem. Res. 42, 938-947 (2009).</li><li>Le&#244;n-Rodr&#237;guez, L. M. D., Lubag, A. J. M., Malloy, C. R., Martinez, G. V., Gillies, R. J., Sherry, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responsive+MRI+agents+for+sensing+metabolism+in+vivo.">Responsive MRI agents for sensing metabolism in vivo.</a> Acc. Chem. Res. 42, 948-957 (2009).</li><li>Castelli, D. D., Gianolio, E., Crich, S. G., Terreno, E., Aime, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+containing+nanosized+systems+for+MR-molecular+imaging+applications.">Metal containing nanosized systems for MR-molecular imaging applications.</a> Coord. Chem. Rev. 252, 2424-2443 (2008).</li><li>Caravan, P., Ellison, J. J., McMurry, T. J., Lauffer, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gadolinium(III)+chelates+as+MRI+contrast+agents:+structure,+dynamics,+and+applications.">Gadolinium(III) chelates as MRI contrast agents: structure, dynamics, and applications.</a> Chem. Rev. 99, 2293-2352 (1999).</li><li>Lauffer, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paramagnetic+metal+complexes+as+water+proton+relaxation+agents+for+NMR+imaging:+theory+and+design.">Paramagnetic metal complexes as water proton relaxation agents for NMR imaging: theory and design.</a> Chem. Rev. 87, 901-927 (1987).</li><li>Yoo, B., Pagel, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+responsive+MRI+contrast+agents+for+molecular+imaging.">An overview of responsive MRI contrast agents for molecular imaging.</a> Front. Biosci. 13, 1733-1752 (2008).</li><li>Pandya, S., Yu, J., Parker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+emissive+europium+and+terbium+complexes+for+molecular+imaging+and+sensing.">Engineering emissive europium and terbium complexes for molecular imaging and sensing.</a> Dalton Trans. 23, 2757-2766 (2006).</li><li>Nwe, K., Xu, H., Regino, C. A. S., Bernardo, M., Ileva, L., Riffle, L., Wong, K. J., Brechbiel, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+in+the+preparation+of+dendrimer-based+bifunctional+diethylenetriaminepentaacetic+acid+MR+contrast+agent+derivatives.">A new approach in the preparation of dendrimer-based bifunctional diethylenetriaminepentaacetic acid MR contrast agent derivatives.</a> Bioconjugate Chem. 20, 1412-1418 (2009).</li><li>Nwe, K., Bernardo, M., Regino, C. A. S., Williams, M., Brechbiel, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+MRI+properties+between+derivatized+DTPA+and+DOTA+gadolinium-dendrimer+conjugates.">Comparison of MRI properties between derivatized DTPA and DOTA gadolinium-dendrimer conjugates.</a> Bioorg. Med. Chem. 18, 5925-5931 (2010).</li><li>Caravan, P., Das, B., Deng, Q., Dumas, S., Jacques, V., Koerner, S. K., Kolodziej, A., Looby, R. J., Sun, W. -. C., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lysine+walk+to+high+relaxivity+collagen-targeted+MRI+contrast+agents.">A lysine walk to high relaxivity collagen-targeted MRI contrast agents.</a> Chem. Commun. , 430-432 (2009).</li><li>Le&#244;n-Rodr&#237;guez, L. M. D., Kovacs, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+synthesis+and+chelation+chemistry+of+DOTA-peptide+conjugates.">The synthesis and chelation chemistry of DOTA-peptide conjugates.</a> Bioconjugate Chem. 19, 391-402 (2008).</li><li>Boswell, C. A., Eck, P. K., Regino, C. A. S., Bernardo, M., Wong, K. J., Milenic, D. E., Choyke, P. L., Brechbiel, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+characterization,+and+biological+evaluation+of+integrin+&#945;V&#946;3-targeted+PAMAM+dendrimers.">Synthesis, characterization, and biological evaluation of integrin &#945;V&#946;3-targeted PAMAM dendrimers.</a> Mol. Pharm. 5, 527-539 (2008).</li></ol>

No conflicts of interest declared.

As the only unique feature to Tregs, suppressive function should be tested reliably and uniformly between subjects at different phases of disease development within the same and between different studies. We offer details of the suppression assay developed in our laboratory as our contribution to the standardization of this assay. In our extensive optimization study, we have determined that T cell stimulation with anti-human CD3-coated beads (UCHT1 clone, in concentration of 1&mu;g/ml (as opposed to commercially availabl...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent or instrument</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments (optional)</th>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>Amersham Pharmacia Biotech</td>
			<td>17-1440-03</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DPBS-1X</td>
			<td>Gibco </td>
			<td>14190-144</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Invitrogen</td>
			<td>15250-061</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>anti-CD4 microbeads</td>
			<td>Miltenyi</td>
			<td>130-045-101</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pre-separation filters</td>
			<td>Miltenyi</td>
			<td>130-041-407</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LS column</td>
			<td>Miltenyi</td>
			<td>130-042-401</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Invitrogen</td>
			<td>15575-020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>B4287</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-human CD4-APCCy7 (clone RPA-T4)</td>
			<td>BD Pharmingen</td>
			<td>557852</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-human CD25-PE (clone M-A251; IL-2R&alpha;)</td>
			<td>BD Pharmingen</td>
			<td>555432</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-human CD8-FITC (clone RPA-T8)</td>
			<td>BD Pharmingen</td>
			<td>555366</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-human CD14-FITC (clone M5E2; LPS receptor)</td>
			<td>BD Pharmingen</td>
			<td>555397</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-human CD32-FITC (clone FLI8.26; Fc&gamma;R-type II)</td>
			<td>BD Pharmingen</td>
			<td>555448</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-human CD116-FITC (clone M5D12; GM-CSFR&alpha; chain)</td>
			<td>BD Pharmingen</td>
			<td>554532</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dynalbeads M-450 tosylactivated</td>
			<td>Invitrogen</td>
			<td>140-13</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-human CD3</td>
			<td>Ancell</td>
			<td>144-024</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Buffer1</td>
			<td>Homemade</td>
			<td>&nbsp;</td>
			<td>0.1M Na2B4O7 pH7.6</td>
		</tr>
		<tr>
			<td>Buffer2</td>
			<td>Homemade</td>
			<td>&nbsp;</td>
			<td>PBS/2mM EDTA/ 0.1% BSA pH7.4</td>
		</tr>
		<tr>
			<td>Buffer3</td>
			<td>Homemade</td>
			<td>&nbsp;</td>
			<td>0.2M Tris/0.1% BSA pH8.5</td>
		</tr>
		<tr>
			<td>Complete RPMI media</td>
			<td>Homemade</td>
			<td>&nbsp;</td>
			<td>RPMI 1640 media 
2 mM L-glutamine 
5 mM HEPES 
100 U/&mu;g/ml peni/strept 
0.5 mM sodium pyruvate
</td>
		</tr>
		<tr>
			<td>[3H] thymidine</td>
			<td>Perkin Elmer</td>
			<td>NET027Z005MC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>human pooled AB serum </td>
			<td>Atlanta Biologicals</td>
			<td>S40110</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Multiscreen harvest plate</td>
			<td>Millipore</td>
			<td>MAHFC1H60</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscint 20</td>
			<td>Perkin Elmer</td>
			<td>6013621</td>
			<td>&nbsp;</td>
		</tr>
		
	</tbody>
</table>

human in vitro suppression as screening tool for the recognition of an early state of immune imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Watch this Scientific Journal Video about Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance at JoVE.com

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

human-vitro-suppression-as-screening-tool-for-recognition-an-early

Research

JoVE Journal

Immunology and Infection

14.4K Views.  Medical College of Wisconsin. Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Video: Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

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. ...

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...