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.

Methylation of cytosines by DNA methyltransferases allows for the epigenetic control of transcription at many genes. The INS gene in humans is almost exclusively expressed in islet &#946; cells, and there appears to be a correlation between the frequency of methylation of cytosines in the INS gene to silencing of its transcription11. As such, most cell types show substantially higher frequencies of methylation of the INS gene at various cytosines compared to &#946; cells11...

Type 1 diabetes (T1D) is an autoimmune disease that is characterized by the destruction of insulin-producing islet &#946; cells by autoreactive T cells1. The diagnosis of T1D is typically made upon measurement of hyperglycemia (blood glucose &#62; 200 mg/dl) in a lean, young individual, who might present with ketoacidosis as evidence of insulin deficiency. At the time of diagnosis of T1D, there is evidence for substantial loss of &#946; cell function and mass (from 50 - 90%)2. In clinical studies, several immune modulatory drugs that were instituted at the time of diagnosis resulted in the stabilization of &#946; cell function (and presumably mass), but none have resulted in clinical remission of disease, a finding that has raised the call for the development of biomarkers for earlier detection of the disease and for the longitudinal tracking of effectiveness of combination therapies3,4. Efforts by international consortia, such as the Human Islet Research Network Consortium at the National Institutes of Heath5, have emphasized the need to develop biomarkers that focus on &#946; cell stress and death in T1D.
In line with these efforts, our group and others have recently developed biomarker assays that measure circulating, epigenetically modified DNA fragments that arise primarily from dying &#946; cells6-9. In all of the published assays to date, the focus has been on the quantitation of the human gene encoding preproinsulin (INS), which demonstrates greater degrees of unmethylated CpG sites in the coding and promoter regions compared to other cell types. The liberation of unmethylated INS DNA fragments was hypothesized as arising primarily from dying (necrotic, apoptotic) &#946; cells. Our recent studies showed that in youth, elevations in both unmethylated and methylated INS DNA at position -69 bp (relative to the transcriptional start site) were observed in new-onset T1D, and together served as specific biomarkers for this population6. These biomarker assays involve the isolation of cell-free DNA from serum or plasma using commercial spin kits, followed by a bisulfite conversion of the isolated DNA (to convert non-methylated cytosines to uracils, leaving methylated cytosines intact).
In this report, we describe the technical aspects of serum sample collection, isolation of cell-free DNA from serum, bisulfite conversion, and performance of droplet digital PCR (henceforth, digital PCR) for differentially methylated 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.

To interpret data appropriately, we use plasmid controls for both the unmethylated and methylated target INS DNA in each digital PCR run. These controls ensure that signals corresponding to methylated and unmethylated DNA are clearly distinguishable. Figure 1 shows the 2-D scatter plots corresponding to droplets for plasmid controls containing bisulfite-converted unmethylated INS DNA (Figure 1A), methylated INS DNA (Figu...

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.

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 ...

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6.4K Views.  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. 

Video: 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.

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

Immunology and Infection

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).

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

Biology

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.

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

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.

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

Medicine

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.

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

Bioengineering

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.

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.

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.

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.

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

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.

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

Isolated Pancreatic Islet Treatment and Apoptosis Measurement

This study investigates the effect of pro-inflammatory cytokines on pancreatic islets, particularly insulin-producing &#946;-cells, using a combination of fluorescence staining techniques and confocal microscopy to assess cell viability, apoptosis, and &#946;-cell-specific death. Isolated mouse islets were treated with varying concentrations of a cytokine cocktail, including TNF-&#945;, IL-1&#946;, and IFN-&#947;, to mimic immune-mediated apoptosis during the development of type 1 diabetes. The viability of islet cells was evaluated with FDA/PI dual staining, where FDA conversion to fluorescein indicated viable cells, and PI marked membrane-compromised cells. YOPRO-1 and nuclear staining provided additional data on apoptosis, with Annexin-V confirming early apoptotic cells. Quantitative analysis revealed significant increases in apoptosis and cell death rates in cytokine-treated islets. To specifically assess effects on &#946;-cells, Zn2+ selective indicator staining was used to label insulin-producing cells through the zinc association in insulin granules, revealing substantial &#946;-cell loss following treatment of islets with pro-inflammatory cytokines for 24 h. These multi-staining protocols effectively capture and quantify the extent of cytokine-induced damage in islets and can be used to evaluate therapeutics designed to prevent &#946;-cell apoptosis in early type 1 diabetes.

This study utilizes multi-staining fluorescence-based markers of cell death and apoptosis combined with confocal microscopy to assess cytokine-induced apoptosis and &#946;-cell-specific death in pancreatic islets. It reveals spatial and temporal changes in cell death and apoptosis in response to extracellular stimuli.

This study investigates the effect of pro-inflammatory cytokines on pancreatic islets, particularly insulin-producing &#946;-cells, using a combination of ...

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

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