This work was supported by National Institutes of Health grant UC4 DK104166 (to RGM). We wish to acknowledge the assistance of the Indiana Diabetes Research Center Translation Core supported by National Institutes of Health grant P30 DK097512.

Ethics Statement: Protocols were approved by the Indiana University Institutional Review Board. Parents of subjects provided written informed consent, and children older than 7 years provided assent for their participation.
1. Serum Processing
NOTE: The assay as described has been rigorously tested using human serum isolated as follows.
<ol>
	<li>Collect blood in one red top (no-additive; uncoated) blood collection tube.</li>
	<li>Let sit at room temperature for 30 min to allow the clot to form.</li>
	<li>Centrifuge tube at 2,000 x g for 10 min at room temperature. Transfer the supernatant (serum) to a new tube designated for serum storage (polypropylene screw top tubes, size dependent on samples).</li>
	<li>Store samples at -80 &#186;C until ready for DNA isolation.</li>
</ol>
2. Serum DNA Extraction
NOTE: DNA is extracted with a DNA extraction kit using 50 &#181;l of serum (recommended), following manufacturer&#39;s protocol with some modifications.
<ol>
	<li>Prepare 200 &#181;l lysis buffer + 1 &#181;l poly(A) (5 &#181;g) per sample in an microcentrifuge tube. Vortex for 15 sec. Set aside until Step 2.3.</li>
	<li>Take serum samples from -80 &#186;C freezer and thaw at room temperature. Bring serum sample volume up to 200 &#181;l total using phosphate-buffered saline (PBS, with calcium chloride and magnesium chloride).</li>
	<li>Lyse the sample by adding 20 &#181;l protease (1.4 Anson units/ml) to sample, followed by 200 &#181;l lysis buffer/polyA mix (from Step 2.2) and vortex for 8 sec. Incubate sample at 56 &#176;C for 10 min, then briefly centrifuge for 7 sec at top speed in a microcentrifuge (&#62;10,000 x g).</li>
	<li>Precipitate DNA by adding 230 &#181;l 100% ethanol to sample and vortex for 8 sec. Briefly centrifuge for 7 sec at top speed (&#62;10,000 x g).</li>
	<li>Apply the DNA to the spin column by adding 600 &#181;l sample mixture to mini spin column. Centrifuge at 6,000 x g for 1 min. Discard the flow-through and place column in a clean tube.</li>
	<li>Wash the DNA by adding 500 &#181;l wash buffer 1 to the column. Centrifuge at 6,000 x g for 1 min. Discard the flow-through and place column in a clean tube. Add 500 &#181;l wash buffer 2 to column and centrifuge at top speed (&#62;10,000 x g) for 3 min. Place column in a new 1.5 ml microcentrifuge tube and centrifuge at top speed again for 1 min.</li>
	<li>Elute the DNA by placing column in a new 1.5 ml microcentrifuge tube and adding 60 &#181;l elution buffer directly on the filter. It is important to switch tips between samples to prevent cross contamination. Incubate at room temperature for 5 min, then centrifuge at 6,000 x&#160;g for 1 min. Add another 60&#160;&#956;l of elution buffer and centrifuge at 6,000 x g for 1 min.&#160;</li>
	<li>Store DNA at -20 &#186;C for later use, or proceed immediately to bisulfite conversion.</li>
</ol>
3. Bisulfite Conversion
NOTE: Bisulfite conversion is performed using a bisulfite conversion kit, following the manufacturer&#39;s protocol with some modifications.
<ol>
	<li>To convert unmethylated cytosines to uracils, add 130 &#181;l of bisulfite conversion reagent to 20 &#181;l of DNA from step 2.7 in a PCR tube (single 0.2 ml PCR tube or 8-strip PCR tubes). Save the remaining 40 &#181;l of DNA for future use, or use in replicate reactions. Mix 20 times by pipetting up and down and centrifuge briefly to make sure no drops are on sides or lid. Incubate in a water bath or thermal cycler as follows: 98 &#176;C for 8 min, 54 &#176;C for 60 min, 4 &#176;C for 5 min.
	<ol>
		<li>Proceed to step 3.2 or store at 4 &#176;C for up to 20 hr.</li>
	</ol>
	</li>
	<li>Add 600 &#181;l of binding buffer to spin column and place the column in the provided collection tube. Apply the bisulfited DNA to column and mix by pipetting up and down 10 times. Centrifuge at top speed (&#62;10,000 x g) for 30 sec. Discard flow-through.</li>
	<li>Wash DNA by adding 100 &#181;l of wash buffer to column and centrifuge at top speed (&#62;10,000 x g) for 30 sec.</li>
	<li>Perform desulphonation to remove sulphonate group to finalize the conversion of unmethylated cytosines to uracil by adding 200 &#181;l of desulphonation buffer to the column. Let stand at room temperature for 20 min, then centrifuge at top speed (&#62;10,000 x g) for 30 sec.</li>
	<li>Wash DNA by adding 200 &#181;l of wash buffer to the column and centrifuge at full speed for 30 sec. Discard the flow-through and repeat this wash step.</li>
	<li>Place the column into a 1.5 ml microcentrifuge tube. Elute DNA by adding 10 &#181;l elution buffer directly to column filter. Incubate for 1 min. Centrifuge for 30 s at full speed to elute DNA. Quantify recovered DNA by spectrophotometry at A260. 
	NOTE: The DNA detected by spectrophotometry represents primarily the carrier polyA DNA that was added at step 2.3 above. Typically, recovery of polyA is &#8805;85% of the input (&#62;4.25 &#181;g).</li>
	<li>Store DNA at -20 &#176;C until ready to proceed with digital PCR.</li>
</ol>
4. Multiplex Digital PCR
<ol>
	<li>Make a master mix with enough solution for each sample and control. Include at least one sample containing water (negative control), one sample of a plasmid containing unmethylated INS (1 pg, positive control), one sample of plasmid containing methylated INS (1 pg, positive control), and one sample containing a 1:1 mixture of plasmids.
	<ol>
		<li>To prepare the PCR master mix, add 12.5 &#181;l per reaction of PCR buffer (e.g., 125 &#181;l per 10 samples), 1.25 &#181;l per reaction primer/probe mix (e.g., 12.5 &#181;l per 10 samples), 8.25 &#181;l per reaction water (e.g., 82.5 &#181;l per 10 samples), 0.5 &#181;l per reaction EcoR1 enzyme (e.g., 5 &#181;l per 10 samples). 
		NOTE: Primers and probes used here are as follows: Forward Primer: 5&#39;-GGAAATTGTAGTTTTAGTTTTTAGTTATTTGT-3&#39;; Reverse Primer: 5&#39;-AAAACCCATCTCCCCTACCTATCA-3&#39;; FAM probe: FAM-5&#39;-ACCCCTACCACCTAAC-3&#39;-MGB; VIC probe: VIC-5&#39;-ACCCCTACCGCCTAAC-3&#39;-MGB.</li>
	</ol>
	</li>
	<li>Set up multiplex PCR reaction in a 96-well plate. Add 19.5 &#181;l of master mix to each well. Add 2.5 &#181;l of bisulfite converted DNA sample into each appropriate well (save the remaining 7.5 &#181;l of bisulfite converted DNA or use in replicate reactions). Mix by pipetting up and down several times. 
	NOTE: All 8 wells in a row must contain sample or buffer control.</li>
	<li>Seal with a foil seal using a plate sealer and centrifuge in a plate spinner until there is no liquid on the sides of the wells.</li>
	<li>Set up the automated droplet generator on the touchscreen. 
	NOTE: All 8 wells in the cartridge must contain sample or buffer control.
	<ol>
		<li>Set the number of rows being used by touching the row(s) in which samples are loaded into on the 96-well plate from step 4.2 and highlight the row in blue to indicate an active row.</li>
		<li>Load consumables from back to front to avoid contamination. To start, add cartridges along the back row of the instrument. Load tips into center row of the instrument. 
		NOTE: The cartridges can only fit into the holders in the correct orientation.</li>
		<li>Place 96-well plate into the instrument. Remove a cold block from -20 &#176;C freezer and place a new skirted 96-well plate into it. Place it inside the instrument next to the sample loaded 96-well plate.</li>
		<li>Add oil to dispenser in the front of the instrument. Select type of oil on touchscreen.</li>
		<li>Touch the blue start button to start the run. Confirm plate setup and touch start run button to begin.</li>
	</ol>
	</li>
	<li>Once finished, remove the 96-well plate that contains the newly formed droplets and seal with a foil seal using a plate sealer.</li>
	<li>Perform PCR10 on a thermal cycler using the following program: 95 &#176;C for 10:00 min; 40 cycles at: 94 &#176;C for 00:30 min, 57.5 &#176;C for 01:00 min; 98 &#176;C for 10:00 min; 12 &#176;C for 10 min or up to 24 hr.</li>
	<li>Place the plate on the droplet reader and set up the reader.
	<ol>
		<li>Click on the setup tab. Open a new template by clicking template &#62; new. Enter a file name.</li>
		<li>Use the well editor to set the parameters for each sample. Give each sample a unique name, set experiment to Rare Event Detection (R-RED), and set master mix to the mix used in step 4.1.</li>
		<li>Give Target 1 a name (i.e., unmethylated) and set Target 1 as unknown (U). Give Target 2 a name (i.e., methylated) and set Target 2 as reference (R).</li>
		<li>Click Run tab to start the run. In the run options window, select the detection chemistry (FAM/VIC).</li>
	</ol>
	</li>
</ol>
5. Data Analysis
<ol>
	<li>Open results in analyze tab and analyze using 2-D plot based on positive controls (see Figure 1).</li>
	<li>Export CSV file generated by droplet reader to a spreadsheet program for data analysis.</li>
	<li>To generate &#34;copies per &#181;l&#34; of serum, use the formula: (concentration*250)/(volume of serum used for DNA isolation in &#181;l).6 
	NOTE: The data is typically converted to log10 when acquired from human serum to ensure normal distribution. Alternatively, data can be plotted on a log10 scale and analyzed by non-parametric statistics.</li>
</ol>
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<ol>
	<li>Lehuen, A., Diana, J., Zaccone, P., Cooke, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cell+crosstalk+in+type+1+diabetes.">Immune cell crosstalk in type 1 diabetes.</a> Nat. Rev. Immunol. 10 (7), 501-513 (2010).</li><li>Sherry, N. A., Tsai, E. B., Herold, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+history+of+beta-cell+function+in+type+1+diabetes.">Natural history of beta-cell function in type 1 diabetes.</a> Diabetes. 54, 32-39 (2005).</li><li>Maganti, A., Evans-Molina, C., Mirmira, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+immunobiology+to+&#946;-cell+biology:+the+changing+perspective+on+type+1+diabetes.">From immunobiology to &#946;-cell biology: the changing perspective on type 1 diabetes.</a> Islets. 6 (2), 28778 (2014).</li><li>Matthews, J. B., Staeva, T. P., Bernstein, P. L., Peakman, M., von Herrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ITN-JDRF+Type+1+Diabetes+Combination+Therapy+Assessment+Group+Developing+combination+immunotherapies+for+type+1+diabetes:+recommendations+from+the+ITN-JDRF+Type+1+Diabetes+Combination+Therapy+Assessment+Group.">ITN-JDRF Type 1 Diabetes Combination Therapy Assessment Group Developing combination immunotherapies for type 1 diabetes: recommendations from the ITN-JDRF Type 1 Diabetes Combination Therapy Assessment Group.</a> Clin. Exp. Immunol. 160 (2), 176-184 (2010).</li><li>. Human Islet Research Network | HIRN Available from: <a target="_blank" href="https://hirnetwork.org/">https://hirnetwork.org/</a> (2016)</li><li>Fisher, M. M., Watkins, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevations+in+Circulating+Methylated+and+Unmethylated+Preproinsulin+DNA+in+New-Onset+Type+1+Diabetes.">Elevations in Circulating Methylated and Unmethylated Preproinsulin DNA in New-Onset Type 1 Diabetes.</a> Diabetes. 64 (11), 3867-3872 (2015).</li><li>Husseiny, M. I., Kaye, A., Zebadua, E., Kandeel, F., Ferreri, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-Specific+Methylation+of+Human+Insulin+Gene+and+PCR+Assay+for+Monitoring+Beta+Cell+Death.">Tissue-Specific Methylation of Human Insulin Gene and PCR Assay for Monitoring Beta Cell Death.</a> PLoS ONE. 9 (4), 94591 (2014).</li><li>Herold, K. C., Usmani-Brown, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;+cell+death+and+dysfunction+during+type+1+diabetes+development+in+at-risk+individuals.">&#946; cell death and dysfunction during type 1 diabetes development in at-risk individuals.</a> J. Clin. Invest. 125 (3), 1163-1173 (2015).</li><li>Lehmann-Werman, R., Neiman, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+tissue-specific+cell+death+using+methylation+patterns+of+circulating+DNA.">Identification of tissue-specific cell death using methylation patterns of circulating DNA.</a> Proc. Natl. Acad. Sci. U.S.A. 113 (13), 1826-1834 (2016).</li><li>Saiki, R. K., Gelfand, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer-directed+enzymatic+amplification+of+DNA+with+a+thermostable+DNA+polymerase.">Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.</a> Science. 239 (4839), 487-491 (1988).</li><li>Kuroda, A., Rauch, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+gene+expression+is+regulated+by+DNA+methylation.">Insulin gene expression is regulated by DNA methylation.</a> PloS One. 4 (9), 6953 (2009).</li><li>Akirav, E. M., Lebastchi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+&#946;+cell+death+in+diabetes+using+differentially+methylated+circulating+DNA.">Detection of &#946; cell death in diabetes using differentially methylated circulating DNA.</a> Proc. Natl. Acad. Sci. U.S.A. 108 (47), 19018-19023 (2011).</li><li>Fisher, M. M., Perez Chumbiauca, C. N., Mather, K. J., Mirmira, R. G., Tersey, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+islet+&#946;-cell+death+in+vivo+by+multiplex+PCR+analysis+of+differentially+methylated+DNA.">Detection of islet &#946;-cell death in vivo by multiplex PCR analysis of differentially methylated DNA.</a> Endocrinology. 154 (9), 3476-3481 (2013).</li><li>Patterson, K., Molloy, L., Qu, W., Clark, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation:+bisulphite+modification+and+analysis.">DNA methylation: bisulphite modification and analysis.</a> J. Vis. Exp. (56), (2011).</li><li>Derks, S., Lentjes, M. H. F. M., Hellebrekers, D. M. E. I., de Bru&#239;ne, A. P., Herman, J. G., van Engeland, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylation-specific+PCR+unraveled.">Methylation-specific PCR unraveled.</a> Cell. Oncol. 26 (5-6), 291-299 (2004).</li><li>Husseiny, M. I., Kuroda, A., Kaye, A. N., Nair, I., Kandeel, F., Ferreri, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+quantitative+methylation-specific+polymerase+chain+reaction+method+for+monitoring+beta+cell+death+in+type+1+diabetes.">Development of a quantitative methylation-specific polymerase chain reaction method for monitoring beta cell death in type 1 diabetes.</a> PloS One. 7 (10), 47942 (2012).</li><li>Hindson, B. J., Ness, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Droplet+Digital+PCR+System+for+Absolute+Quantitation+of+DNA+Copy+Number.">High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number.</a> Anal. Chem. 83 (22), 8604-8610 (2011).</li><li>Sozzi, G., Roz, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+prolonged+storage+of+whole+plasma+or+isolated+plasma+DNA+on+the+results+of+circulating+DNA+quantification+assays.">Effects of prolonged storage of whole plasma or isolated plasma DNA on the results of circulating DNA quantification assays.</a> J. Natl. Cancer Inst. 97 (24), 1848-1850 (2005).</li></ol>

The authors have nothing to disclose.

Die Methylierung von Cytosin durch DNA-Methyltransferasen ermöglicht die epigenetische Kontrolle der Transkription bei vielen Genen. Das INS - Gen beim Menschen wird fast ausschließlich in islet β - Zellen exprimiert, und es scheint Silencing seiner Transkriptions 11 eine Korrelation zwischen der Häufigkeit der Methylierung von Cytosin in dem INS - Gen zu sein. Als solches zeigen die meisten Zelltypen wesentlich höhere Frequenzen der Methylierung des INS - Gen an verschiedenen Cytosine verglichen Zellen 11 zu &amp; beta; - 13. Es wurde vorgeschlagen , dass die Prävalenz von β Zelltod durch Analyse der Serumspiegel von zellfreien unmethylierte DNA INS überwacht werden könnte, die weitgehend aus β sterbende Zellen ergeben würden, die ihre DNA 12 befreien. Die Bisulfit - Reaktion wandelt unmethylierte Cytosine in urazile, methylierte Cytosine unverändert 14 zu verlassen. Die Sequenzierung oder PCR-Analyse of Bisulfit-umgewandelten DNA erlaubt daher die Bestimmung, ob der ursprüngliche Cytosin methylierte oder nicht. Methylierungs-spezifische PCR unter Verwendung von Farbstoff-basierte Technologie nutzt Unterschiede in PCR - Effizienz durch 3 &#39;Basenpaar - Mismatches zwischen Primern und Template, und ermöglicht dadurch , dass Unterscheidung von nicht - methylierten gegen methylierte Cytosine folgende Bisulfitumwandlung von DNA 15. Dementsprechend wurde auf Farbstoffbasis PCR verwendet , um das relative Verhältnis von unmethylierten zu methyliert INS in frühen Studien von T1D aussetzt 12,16 zu quantifizieren. Jedoch farbstoffbasierte PCR - Technologie hat mehrere Nachteile, einschließlich begrenzte Spezifität, fehlende absolute Quantifizierung von DNA - Fragment - Konzentrationen, und die Unfähigkeit , zu multiplexieren (dh detektieren sowohl methylierte und unmethylierte DNA - Spezies in der gleichen Reaktion). Andere Gruppen haben die direkte Sequenzierung der zellfreien DNA-Fragmente durchgeführt wird, eine Technik, die große Überwachung mehrerer differentiell methylierte CpG-Stellen mit viel ermöglichter Spezifität 9. Jedoch erfordert die Sequenzierungstechnik große Probenvolumina ist nicht leicht zugänglich unverbrauchten Proben zu verwenden, und ist teuer. Als Folge dieser Einschränkungen unserer Gruppe und andere haben digitale PCR verwendet wird, eine Technik, die absolute Quantifizierung von DNA-Fragmenten stellt nur Mikrolitermengen von Serum aus frischem oder überhöht Proben erfordert, zugänglich ist zum Multiplexen, zeigt eine höhere Empfindlichkeit als herkömmliche quantitative PCR und ist kostengünstiger als die direkte Sequenzierung. Digital-PCR-Technologie umfasst die Verwendung von herkömmlichen Primer / Sonden-Assays und eine Mikrofluidik-basierte Partitionierung einer Wasser-in-Öl-PCR-Reaktion in ~ 20.000 Tröpfchen. thermische Zyklen der partitionierten Reaktions folgenden werden die Tröpfchen anschließend von einem Durchflusszytometer analysiert, um zu identifizieren, Tröpfchen mit positiven und negativen Signale. Poisson-Statistik wird verwendet um absolute Mengen zu berechnen (Kopienzahlen) jeder DNA-Spezies. Die konzeptionelle details der digitalen PCR wurden an anderer Stelle 17 beschrieben. Dieses Protokoll beschreibt die Messung von β Zelltod in Menschen durch differentiell methylierte DNA INS durch digitale PCR gemessen wird . Unsere hier präsentierten Daten und anderswo 6 zeigen , dass Primer / Sonde - Kombinationen , die Differential - Methylierung von Cytosin an Position -69 (zu der Transkriptionsstartstelle des INS relativ) erfassen für eine ausreichende Spezifität aufweisen , entweder die nicht methylierten Cytosin (FAM - Sonde) oder der methylierte Cytosin ( VIC - Sonde) (siehe Abbildung 1A und B). Mischungen von Plasmiden sowohl methylierte und unmethylierte INS - Fragmente an der Position -69 bp (dh Bisulfit-umgewandelten DNA mit entweder Uracil oder Cytosin an Position -69 bp) zeigen Tröpfchen , die eine oder die andere Plasmid oder beide Plasmide (double-positive Signale enthält , in Abbildung 1C). Die Poisson statistische Berechnung nutzt the Tröpfchen Zahlen, die für eine gegebene Sonde positiv sind (ob Single-positive oder doppelt positiven), um die Kopienzahl jedes methylierte oder unmethylierte DNA-Fragment abzuleiten. Wenn für die Messung von differentiell methylierte Cytosine Humanserum unter Verwendung von an Position -69 bp in zellfreien DNA (Abbildung 3), zeigen unsere Daten zwei wichtige Funktionen: (1) Ebenen von methylierter INS sind 5-10-fach höher in diesen Fächern als nicht - methylierten INS (unabhängig von der Diagnose), und (2) absolute Pegel beider unmethylierte und methylierte INS sind höher bei Patienten mit T1D neu einsetzende Vergleich zu den Kontrollen. Während die höheren Ebenen von methylierter INS im Vergleich zu nicht - methylierten INS wahrscheinlich die größere Last der zellfreien DNA ausgehend von nicht-β - Zellen reflektieren, reflektiert die höheren Ebenen der beiden Arten von INS in neu einsetzende T1D Themen , die sowohl für eine Erhöhung der vorherrschenden β Zelltod (unmethylierte INS), und möglicherweise den Tod / Umsatz von anderen Zelltypen , die mit dem Krankheitszustand assoziiert sind (zB angeborenen und adaptiven Immunzellen). Weitere Spekulationen auf den Erhebungen der beiden Arten von INS in neu einsetzende T1D Probanden wurde bisher 6 diskutiert. Unabhängig von den Quellen der beiden Arten von INS in Serum von diesen Personen ist es bemerkenswert , dass die Messung der Verhältnisse von unmethylierten zu methyliert INS (wie durch andere Forscher berichteten unter Verwendung farbstoffbasierte PCR) nicht die signifikanten Unterschiede in absoluten Niveaus aufgenommen haben hier beschriebenen und in unserer jüngsten Veröffentlichung 6, so dass für diese Art von Biomarkeranalyse die Macht der digitalen PCR betont. Einige Vorsichtsmaßnahmen und Einschränkungen ist zu beachten,. Wir glauben, es ist wichtig, dass das Blut innerhalb von 4 Stunden der Entnahme verarbeitet wird und entweder sofort für die DNA-Isolierung verwendet oder bei -80 ° C für die zukünftige Verwendung gespeichert. In nicht publizierten Studien haben wir observed der Sammlung einen gewissen Verlust an DNA Erholung nach 4 Stunden, möglicherweise an DNA - Abbau aufgrund, wie in 18 Studien der Langzeitlagerung von Proben gesehen. Aufgrund der erhöhten Empfindlichkeit der digitalen PCR, sollte besondere Sorgfalt Kontamination der Proben vermieden wird, die sonst nicht in der traditionellen qPCR bemerkbar wäre. Schließlich ist eine zentrale Einschränkung der Technik der Analyse von Proben, bei denen Tröpfchenerzeugung nicht ~ 10.000 Tröpfchen nicht übersteigt. Solche Proben bieten keine zuverlässige Poisson-Statistik, und als solche kann nicht interpretierbar betrachtet werden. Mehrere Ursachen solcher geringen Tropfenerzeugung könnten technische Fehler bei der Probenhandhabung, technische Probleme mit dem Tropfengenerator umfassen, oder Fragen im Zusammenhang mit der Qualität der verwendeten Reagenzien. Zusammenfassend beschreibt dieser Bericht die detaillierten Protokoll für die Messung von differentiell methyliert, zellfreien INS DNA an Position -69 bp durch digitale PCR. Das hier beschriebene Protokoll kann auf jede angepasst werdenDNA-Spezies, für die Detektion von differentiell methylierte Cytosine gewünscht wird, ob aus dem Verkehr oder von isolierten Zellen und Geweben, und absolute Quantifizierung von DNA-Fragmenten zur Verfügung stellen kann.

Typ - 1 - Diabetes (T1D) ist eine Autoimmunerkrankung , die durch die Zerstörung der Insulin produzierenden Inselzellen β - Zellen durch autoreaktive T - Zellen 1 gekennzeichnet ist. Die Diagnose von T1D ist in der Regel bei der Messung von Hyperglykämie (Blutzucker&gt; 200 mg / dl) in einem schlanken, jungen Person gemacht, die mit einer Ketoazidose als Beweis für Insulinmangel darstellen könnten. Zum Zeitpunkt der Diagnose von T1D gibt es Hinweise für einen wesentlichen Verlust von β - Zellfunktion und Masse (50-90%) 2. In klinischen Studien wurden mehrere immunmodulatorischen Medikamente, die zum Zeitpunkt der Diagnose eingeleitet wurden führte zur Stabilisierung der β-Zellfunktion (und vermutlich Masse), aber keine haben in klinischer Remission der Erkrankung führte, eine Feststellung, dass die Forderung nach der Entwicklung erhöht hat von Biomarkern für frühere Erkennung der Krankheit und für die Längs Verfolgung der Wirksamkeit von Kombinationstherapien 3,4. Die Bemühungen der internationalen Konsortien, eine solchedas Human Islet Research Network Consortium s an den National Institutes of Heath 5, haben die Notwendigkeit zur Entwicklung von Biomarkern betont , dass in T1D auf β Zellstress und Tod konzentrieren. Im Einklang mit diesen Bemühungen haben unsere Gruppe und andere kürzlich Biomarker - Assays entwickelt, die epigenetisch modifizierte DNA - Fragmente messen zirkulierenden , die von sterbenden β - Zellen 6 in erster Linie entstehen - 9. In allen veröffentlichten Assays bisher hat sich der Schwerpunkt auf die Quantifizierung des humanen kodierenden Gens Präproinsulin (INS) gewesen, der größer Grade unmethylierte CpG - Stellen in den Codierungs- und Promotorregionen im Vergleich zu anderen Zelltypen veranschaulicht. Die Befreiung von nicht - methylierten INS DNA - Fragmente wurde die Hypothese aufgestellt , als in erster Linie vom Sterben (nekrotischen, apoptotischen) β - Zellen entstehen. Unsere jüngsten Studien haben gezeigt , dass in der Jugend, Erhöhungen in sowohl unmethylierte und methylierte DNA INS an Position -69 bp (bezogen auf ter Transkriptionsstartstelle) wurden in neu einsetzende T1D beobachtet und diente zusammen als spezifische Biomarker für diese Population 6. Diese Biomarker Assays beinhalten die Isolierung von zellfreien DNA aus Serum oder Plasma unter Verwendung handelsüblicher Kits Spin, gefolgt von einer Bisulfit-Umwandlung von der isolierten DNA (an nicht-methylierte Cytosine bis uracile konvertieren, so dass methylierte Cytosine intakt). In diesem Bericht beschreiben wir die technischen Aspekte der Serumprobe Sammlung, Isolierung von zellfreien DNA aus Serum, Bisulfitumwandlung und Leistung von Tröpfchen digitalen PCR (von nun an, digitale PCR) für differentiell methyliert INS DNA.

<table><tbody><tr><td>Red Top Vacutainer&amp;nbsp;</td><td>Beckon Dickinson</td><td>366441</td><td>no additive, uncoated interior, 10 ml</td></tr><tr><td>Cryovial Tube</td><td>Simport</td><td>T310-3A</td><td>polypropylene, screw cap tube, any size</td></tr><tr><td>QIAamp DNA Blood Mini Kit</td><td>Qiagen</td><td>51106</td><td></td></tr><tr><td>Poly(A)</td><td>Sigma</td><td>P9403</td><td>disloved in TE buffer (10 mM Tris-Cl pH 8.0 + 1 mM EDTA) to 5 &amp;micro;g/&amp;micro;l&amp;nbsp;</td></tr><tr><td>Absolute Ethanol (200 Proof)</td><td>Fisher Scientific</td><td>BP2818-500</td><td></td></tr><tr><td>DPBS (with CaCl&lt;sub&gt;2&lt;/sub&gt; and MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma</td><td>D8662</td><td></td></tr><tr><td>0.2 ml PCR 8-strip Tubes</td><td>MidSci</td><td>AVST</td><td></td></tr><tr><td>8-strip Caps, Dome</td><td>MidSci</td><td>AVSTC-N</td><td></td></tr><tr><td>EZ DNA Methylation-Lightning Kit</td><td>Zymo</td><td>D5031</td><td></td></tr><tr><td>ddPCR Supermix for Probes (No dUTP)</td><td>Biorad</td><td>1863024</td><td></td></tr><tr><td>Buffer Control for Probes</td><td>Biorad</td><td>1863052</td><td></td></tr><tr><td>Human Unmethylated/Methylated Primer/Probe mix</td><td>Life Technologies</td><td>AH21BH1</td><td></td></tr><tr><td>EcoR1</td><td>New England Biolabs</td><td>R0101L</td><td></td></tr><tr><td>twin.tec PCR Plate 96, semi-skirted</td><td>Eppendorf</td><td>951020346</td><td></td></tr><tr><td>Pierceable Foil Heat Seal</td><td>Biorad</td><td>1814040</td><td></td></tr><tr><td>PX1 PCR Plate Sealer</td><td>Biorad</td><td>1814000</td><td></td></tr><tr><td>QX200 AutoDG Droplet Digital PCR System</td><td>Biorad</td><td>1864101</td><td></td></tr><tr><td>Automated Droplet Generation Oil for Probes</td><td>Biorad</td><td>186-4110</td><td></td></tr><tr><td>DG32 Cartridge for Automated Droplet Generator</td><td>Biorad</td><td>186-4108</td><td></td></tr><tr><td>Pipet Tips for Automated Droplet Generator</td><td>Biorad</td><td>186-4120</td><td></td></tr><tr><td>Pipet Tip Bins for Automated Droplet Generator</td><td>Biorad</td><td>186-4125</td><td></td></tr><tr><td>C1000 Touch Thermal Cycler</td><td>Biorad</td><td>1851197</td><td></td></tr><tr><td>QX200 Droplet Reader</td><td>Biorad</td><td>186-4003</td><td></td></tr><tr><td>ddPCR Droplet Reader Oil</td><td>Biorad</td><td>186-3004</td><td></td></tr></tbody></table>

measurement of differentially methylated ins dna species in human serum samples as a biomarker of islet &#946; cell death

The death of islet &#946; cells is thought to underlie the pathogenesis of virtually all forms of diabetes and to precede the development of frank hyperglycemia, especially in type 1 diabetes. The development of sensitive and reliable biomarkers of &#946; cell death may allow for early therapeutic intervention to prevent or delay the development of diabetes. Recently, several groups including our own have reported that cell-free, differentially methylated DNA encoding preproinsulin (INS) in the circulation is correlated to &#946; cell death in pre-type 1 diabetes and new-onset type 1 diabetes. Here, we present a step-by-step protocol using digital PCR for the measurement of cell-free INS DNA that is differentially methylated at cytosine at position -69 bp (relative to the transcriptional start site). We demonstrate that the assay can distinguish between methylated and unmethylated cytosine at position -69 bp, is linear across several orders of magnitude, provides absolute quantitation of DNA copy numbers, and can be applied to samples of human serum from individuals with new-onset type 1 diabetes and disease-free controls. The protocol described here can be adapted to any DNA species for which detection of differentially methylated cytosines is desired, whether from circulation or from isolated cells and tissues, and can provide absolute quantitation of DNA fragments.

Um Daten in geeigneter Weise zu interpretieren, verwenden wir Plasmid - Kontrollen sowohl für die unmethylierte und methylierte Ziel INS DNA in jeder digitalen PCR - Lauf. Diese Kontrollen stellen sicher, dass Signale an methylierter und nicht methylierter DNA entsprechen, sind klar zu unterscheiden. Abbildung 1 zeigt die 2-D Streudiagramme zu Tröpfchen für Plasmid Kontrollen enthält Bisulfit umgewandelt unmethylierte INS DNA (1A) entspricht, methylierte INS DNA (1B) und eine 1: 1 - Gemisch der zwei Plasmide (Figur 1C). Plasmid unmethylierte INS enthält , wird durch die Tröpfchen positiv für die 6-Carboxyfluorescein (FAM) Signal angezeigt, während Plasmid INS durch die Tröpfchen positiv für das Signal VIC angezeigt wird methyliert enthält. In der 1: 1-Plasmid-Gemisch wurde eine Population doppelsträngiger positive Tröpfchen ist zu sehen, entsprechend Tröpfchen, die sowohl ArtenDNA. Zur Unterscheidung positive Tröpfchen aus negativen Tröpfchen werden Daten gated diese 2-D-Plots verwenden. Man beachte , daß in Figur 1A, gibt es eine leichte Verschiebung der FAM-positive Tröpfchen in die VIC Kanal, das anzeigt , dass die Sonde für die methylierte DNA INS weist gewisse Kreuzreaktivität für die unmethylierte DNA INS. Ebenso in Figur 1B, gibt es eine sehr geringe Verschiebung der VIC-positive Tröpfchen in der FAM Kanal anzeigt Kreuzreaktivität der Sonde für die unmethylierte DNA mit methylierter DNA. Ein wesentlicher Vorteil der digitalen PCR ist es immer noch spezifische Signale selbst unterscheiden kann, wenn Sonden nicht zu 100% spezifisch sind. Für eine entsprechende Poisson statistische Berechnungen durch die Software, sollte jeder PCR-Reaktion in mindestens 10.000 Gesamt Tröpfchen aufzuteilen, zeigen eine Gruppe von negativen Tröpfchen, und zeigen eine deutliche Amplitudendifferenz zwischen negativen und positiven Tröpfchen (für eine sichere Gating). Um zu demonstrieren, Linearität unserer Primer, wir performed Mischungen der beiden Plasmide über mehrere Größenordnungen. Wie in Figur 2 gezeigt, können variierende Konzentrationen eines Plasmid linear in der Gegenwart einer konstanten Menge des zweiten Plasmids nachgewiesen werden. Für neue Labore dieses Verfahren durchführen, empfehlen wir eine ähnliche Mischung Experiment konstruieren, um sicherzustellen, dass der Test in einem linearen Detektions Mode arbeitet. Als nächstes wir Serum von drei Patienten mit neu aufgetretenem T1D erhalten (innerhalb von 2 Tagen nach der Diagnose) und drei Kontrollpersonen ohne T1D (Tabelle 1 für die klinische Merkmale sehen). Protokolle wurden von der Indiana University Institutional Review Board genehmigt. Die Eltern der Probanden eine schriftliche Einwilligung informiert. Abbildung 3 zeigt die Quantifizierung von nicht - methylierten und methylierten INS DNA - Kopienzahlen / ul Serum, umgerechnet 10 von diesen Kontrollen und T1D Themen zu protokollieren. Die Daten zeigen, dass i<html> ndividuals mit T1D zeigen erhöhte Werte von sowohl methylierter und nicht methylierter DNA INS im Vergleich zu Kontrollen, ähnlich wie Daten , die zuvor 6 gemeldet. <img alt="Abbildung 1" src="/files/ftp_upload/54838/54838fig1.jpg" /> Abbildung 1: Repräsentative 2-D - Plots von Plasmid - Kontrollen. Plasmid - Standards wurden erzeugt durch Fragmente von Bisulfit-umgewandelt INS DNA beherbergen unmethylierte oder methylierte Cytosin an Position -69 bp relativ zum INS Klonen Transkriptions - Seite. Es zeigen 2-D Diagramme Plasmid enthalten unmethylierte (A) und methylierten (B) INS - DNA und für ein 1: 1 - Mischung der zwei Plasmide (C). Die Pfeile kennzeichnen die nicht methylierten, methylierte und unmethylierte + methyliert (Doppel- positiv) INS DNA-haltigen Tröpfchen. FAM = Ziel 1; VIC = Target 2.ref = &quot;http://ecsource.jove.com/files/ftp_upload/54838/54838fig1large.jpg&quot; target = &quot;_ blank&quot;&gt; Bitte hier klicken, um eine größere Version dieser Figur zu sehen. <img alt="Figur 2" src="/files/ftp_upload/54838/54838fig2.jpg" /> Abbildung 2: Die Quantifizierung von DNA - Targets Control Plasmid Mischungen verwenden. Plasmid - Standards geklonten Fragmente von Bisulfit-umgewandelt INS DNA beherbergen unmethylierte oder methylierte Cytosin an Position -69 bp relativ zum INS enthält Website Transkriptions - Start wurden bei den verschiedenen Kombinationen gemischt gezeigt, dann einer digitalen PCR zu multiplexen. Die Figur zeigt die Quantifizierung der Ziel-DNA-Fragmente, als Kopien / &amp; mgr; l dargestellt; r 2 = 0,989 für unmethylierte DNA INS und r 2 = 0,998 für methylierte DNA INS. <a href="http://ecsource.jove.com/files/ftp_upload/54838/54838fig2large.jpg" target="_blank">Bitte klicken Sie hier</a> , <a href="http://ecsource.jove.com/files/ftp_upload/54838/54838fig2large.jpg" target="_blank">um</a> diesehen eine größere Version dieser Figur. <img alt="Figur 3" src="/files/ftp_upload/54838/54838fig3.jpg" /> Abbildung 3: Die Umlauf Unmethylierte und methylierter INS - DNA - Spiegel in den Kontrollen und Patienten mit T1D. Serumproben wurden von vier Jugendlichen mit neu aufgetretenem T1D gesammelt und von 4 krankheitsfreie, unabhängige Kontrollen, dann verarbeitet für die Messung von differentiell methyliert INS DNA durch digitale PCR. Gezeigt sind Ergebnisse von digitalen PCR Assays für unmethylierte (A) und methylierten (B) INS DNA. Die Daten werden als einzelne Punkte angezeigt und Mittelwert ± SEM. Die Statistiken wurden durch einen zweiseitigen parametrischer Students t - Test analysiert. <a href="http://ecsource.jove.com/files/ftp_upload/54838/54838fig3large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="http://ecsource.jove.com/files/ftp_upload/54838/54838fig3large.jpg" target="_blank">eine größere Version von thi zu sehen</a>Wert. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td> Steuerung </td><td> T1D bei Beginn </td></tr><tr><td> Alter (Jahre) </td><td> 10,3 ± 1,3 </td><td> 9,2 ± 1,0 </td></tr><tr><td> Weiblich männlich </td><td> 1: 3 </td><td> 1: 3 </td></tr><tr><td> BMI Z-Score </td><td> 0,30 ± 0,9 </td><td> 0,74 ± 0,9 </td></tr><tr><td> HbA 1C (%) </td><td></td><td> 11,1 ± 0,6 </td></tr><tr><td> C-Peptid (pmol / L) </td><td></td><td> 285,5 ± 37,0 </td></tr></tbody></table> Tabelle 1: Demographische Daten der Kontrollen und Patienten mit T1D.

Watch this Scientific Journal Video about Measurement of Differentially Methylated INS DNA Species in Human Serum Samples as a Biomarker of Islet Î² Cell Death at JoVE.com

Measurement of Differentially Methylated INS DNA Species in Human Serum Samples as a Biomarker of Islet &#946; Cell Death

Islet &#946; cell death precedes development of type 1 diabetes, and detecting this process may allow for early therapeutic intervention. Here, we provide a detailed description of how to measure differentially methylated INS DNA species in human serum as a biomarker of &#946; cell death.

Die Messung der differentiell methylierter<em&gt; INS</em&gt; DNA-Spezies in Humanserumproben als Biomarker von Islet β Cell Death

The death of islet &#946; cells is thought to underlie the pathogenesis of virtually all forms of diabetes and to precede the development of frank ...

measurement-differentially-methylated-ins-dna-species-human-serum

Nanyang Technological University

Research

JoVE Journal

Genetics

6.4K Aufrufe.  Indiana University School of Medicine. Islet β cell death precedes development of type 1 diabetes, and detecting this process may allow for early therapeutic intervention. Here, we provide a detailed description of how to measure differentially methylated INS DNA species in human serum as a biomarker of β cell death. 

Serie: Measurement of Differentially Methylated INS DNA Species in Human Serum Samples as a Biomarker of Islet β Cell Death

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Methoden zur Beta Cell Death vermittelt durch zytotoxische T-Lymphozyten Beurteilung

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Forschung

Immunologie und Infektion

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

Methylierte DNA Immunpräzipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

Biologie

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to normal and pathological conditions. The goal of this protocol is to generate high-quality, large-scale transcriptome data of each endocrine cell type with the use of a droplet-based microfluidic single-cell RNA sequencing technology. Such data can be utilized to build the gene expression profile of each endocrine cell type in normal or specific conditions. The process requires careful handling, accurate measurement, and rigorous quality control. In this protocol, we describe detailed steps for human pancreatic islets dissociation, sequencing, and data analysis. The representative results of about 20,000 human single islet cells demonstrate the successful application of the protocol.

Here, we present a protocol to generate high-quality, large-scale transcriptome data of single cells from isolated human pancreatic islets using a droplet-based microfluidic single-cell RNA sequencing technology.

Einzellige RNA-Sequenzierung und Analyse menschlicher Pankreas-Inseln

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

Genetik

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Verbessertes Protokoll für Laser Mikrodissektion Of Human Pankreasinseln Von chirurgischen Proben

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Medizin

Sustained Administration of &#946;-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the pancreatic &#946;-cells. In addition, biomaterial particles, hydrogels, and scaffolds also provide a unique opportunity to administer sustained, controllable drug delivery to &#946;-cells in culture and in transplanted tissue models. These technologies allow the study of candidate &#946;-cell proliferation factors using intact islets and a translationally relevant system. Moreover, determining the effectiveness and feasibility of candidate factors for stimulating &#946;-cell proliferation in a culture system is critical before moving forward to in vivo models. Herein, we describe a method to co-culture intact mouse islets with biodegradable compound of interest (COI)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for the purpose of assessing the effects of sustained in situ release of mitogenic factors on &#946;-cell proliferation. This technique describes in detail how to generate PLGA microspheres containing a desired cargo using commercially available reagents. While the described technique uses recombinant human Connective tissue growth factor (rhCTGF) as an example, a wide variety of COI could readily be used. Additionally, this method utilizes 96-well plates to minimize the amount of reagents necessary to assess &#946;-cell proliferation. This protocol can be readily adapted to use alternative biomaterials and other endocrine cell characteristics such as cell survival and differentiation status.

Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of &#946;-cell proliferation. This method is suitable for determining the efficacy of candidate &#946;-cell mitogens.

Sustained Verabreichung von β-Zellen-Mitogene zu Intact Maus Inselchen<em&gt; Ex Vivo</em&gt; Verwendung von biologisch abbaubaren Poly (Milch-co-Glykolsäure) Microspheres

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the ...

Biotechnik

In situ Quantification of Pancreatic Beta-cell Mass in Mice

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic beta-cell proliferation and islet growth is particularly challenging. We have developed a method to capture the distribution of beta-cells in the intact pancreas of transgenic mice with fluorescence-tagged beta-cells with a macro written for ImageJ (rsb.info.nih.gov/ij/). Following pancreatic dissection and tissue clearing, the entire pancreas is captured as a virtual slice, after which the GFP-tagged beta-cells are examined. The analysis includes the quantification of total beta-cell area, islet number and size distribution with reference to specific parameters and locations for each islet and for small clusters of beta-cells. The entire distribution of islets can be plotted in three dimensions, and the information from the distribution on the size and shape of each islet allows a quantitative and qualitative comparison of changes in overall beta-cell area at a glance.

In situ Quantification of Pancreatic Beta-cell Mass in Mice

The following protocol outlines the process of pancreatic dissection for virtual slice imaging, and the subsequent quantification of all GFP-tagged beta-cells in the entire pancreas.

In situ Quantifizierung von Pankreas-Beta-Zellmasse in Mäuse

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic ...

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 &#176;C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 &#176;C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases.

Electrochemiluminescence-Assays für menschliche Insel-Autoantikörper

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

Ein hoher Gehalt<em&gt; In Vitro</em&gt; Pancreatic Islet β-Zell-Replikation-Discovery-Plattform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production

Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those obtained with whole pancreas transplantation. The protocols currently used for human islet isolation are under scrutiny because they are based on the enzymatic digestion of the organ, whereby the pancreas is demolished, its connections to the body are lost and islets are irreversibly damaged. Islet damage is characterized by critical factors such as the destruction of the extracellular matrix (ECM), which represents the 3D framework of the islet niche and whose loss is incompatible with islet euphysiology. Researchers are proposing the use of ECM-based scaffolds derived from the mammalian pancreas to address this problem and ultimately improve islet viability, function, and lifespan. Currently available methods to obtain such scaffolds are harsh because they are largely detergent based. Thus, we propose a new, detergent-free method that creates less ECM damage and can preserve critical components of pancreatic ECM. The results show that the newly developed decellularization protocol allowed the achievement of complete DNA clearance while the ECM components were retained. The ECM obtained was tested for cytotoxicity and encapsulated with human pancreatic islets which showed a positive cellular behavior with insulin secretion when stimulated with glucose challenge. Collectively, we propose a new method for the decellularization of the human pancreas without the use of conventional ionic and non-ionic chemical detergents. This protocol and the ECM obtained with it could be of use for both in vitro and in vivo applications.

<meta charset="utf8"></head><body>Dezellularisierung der humanen Bauchspeicheldrüse ohne Detergenzienz zur Herstellung löslicher extrazellulärer Matrix EZM

The protocol described in this manuscript explains the steps for the fabrication of a soluble extracellular matrix (ECM) from the human pancreas. The solubilized ECM powder obtained through this protocol may be used for the recapitulation of pancreatic islets&#8217; microenvironment in vitro and, potentially, in vivo settings.

Dezellularisierung der humanen Bauchspeicheldrüse ohne Detergenzienz zur Herstellung löslicher extrazellulärer Matrix (ECM)

Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those ...

Die Messung der differentiell methylierter

Zusammenfassung

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Dezellularisierung der humanen Bauchspeicheldrüse ohne Detergenzienz zur Herstellung löslicher extrazellulärer Matrix (ECM)

Dezellularisierung der humanen Bauchspeicheldrüse ohne Detergenzienz zur Herstellung löslicher extrazellulärer Matrix (ECM)