TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Isolated human islets were dispersed into single cells using Accutase (Millipore), ~1500-2000 islet equivalents/ml. 20,000-65,000 islet cells were transferred to individual wells of a 96 well culture plate (CELLSTAR, Greiner Bio-one) for immediate staining for flow cytometry analysis or for culture prior to staining. For intracellular staining, isolated islet cells were fixed in 2% paraformaldehyde (Sigma-Aldrich) and permeabilized using 0.3% saponin (Sigma-Aldrich). The cells were stained with 10E4 mouse anti-human HS mAb (10E4, 1/50; US Biological/Amsbio), mouse anti-mouse Col18 mAb (1/50; Santa Cruz Biotechnol.) or the corresponding isotype control Ig (mouse IgM<sub>κ</sub> or IgG<sub>2bκ</sub>; BD Biosciences) followed by goat anti-mouse Ig-R-PE (1/100; Southern Biotech). The geometric mean fluorescence ratio (GMFR) was calculated by dividing the geometric mean fluorescence intensity (GMFI) of cells stained with primary mAb by the GMFI obtained with the relevant isotype control Ig. Cells were analyzed using a BD LSRI flow cytometer and CellQuest™ Pro software (version 6.0; BD Biosciences).</p>

[BEFORE_START]
<p><strong>Before starting:</strong></p>
<p> </p>
<p>Materials:</p>
<p> </p>
<ol>
<li>Prepare:</li>
</ol>
<p>(i) <u>2% Paraformaldehyde:</u></p>
<p>Add 2 g Paraformaldehyde (#P6148 Sigma) to 50 ml deionised water, a stirrer and 2 drops of 10 M NaOH. Heat at 60°C for 30 mins. Add 40 ml deionised water and 10 ml of 10x PBS and pH to 7.2. Store solution at 4°C.</p>
<p> </p>
<p>(ii)<u> PBS/3 mM EDTA:</u></p>
<p>112 mg EDTA (AJAX #180) in 100 ml PBS, sterile filter using 0.2 μm disposable filter.</p>
<p> </p>
<p>(iii)<u> Beta cell culture medium:</u></p>
<p>RPMI 1640 (Sigma #R0883) 200 ml</p>
<p>Heat-inactivated fetal calf serum (HIFCS) 20 ml</p>
<p>L-Glutamine (Gibco #25030081 200 mM) 2 ml (final 2 mM)</p>
<p>Penicillin G (MP Biomedicals #02194537), 0.06 mg/ml</p>
<p>Streptomycin (Sigma #S9137), 0.10 mg/ml</p>
<p>Neomycin (Sigma #N6386), 0.10 mg/ml</p>
<p> </p>
<p>(iv)<u> PBS/5% HIFCS:</u></p>
<p>500 ml PBS + 25 ml HIFCS</p>
<p> </p>
<p>(v)<u> PBS/5% HIFCS/0.3% saponin:</u></p>
<p>100 ml PBS/5% HIFCS + 300 mg saponin (#S7900, Sigma) and sterile filter with a disposable 0.2μm filter.</p>
<p> </p>
<p>(vi)<u> PBS/5% HIFCS/0.03% saponin:</u></p>
<p>10 ml PBS/5% HIFCS/0.3% saponin + 90 ml PBS/5%HIFCS</p>
<p> </p>
<p> </p>
<ol start="2">
<li>Mabs and pAbs: </li>
</ol>
<p>10E4 (anti-HS) mAb, Amsbio #370255-1</p>
<p>Mouse anti-mouse collagen type XVIII (Col18A1), Santa Cruz Biotechnol #1837-46</p>
<p>IgM<sub>κ</sub> , BD Biosciences #550340</p>
<p>Mouse IgG<sub>2bκ </sub>, BD Biosciences #557351</p>
<p>Goat anti-mouse Ig R-PE, Southern Biotech #1010-09</p>
<p> </p>
<p> </p>
<ol start="3">
<li>Other reagents/materials:</li>
</ol>
<p>Accutase, Millipore #SCR005</p>
<p>Cell culture plates: Cellstar #650180 (Greiner Bio-one)</p>
<p>Cluster tubes, Fisher Biotech #MTS-11C</p>
<p> </p>

[GUIDELINES]
<p>10E4 anti-heparan sulfate (HS) mAb identifies highly sulfated HS localised in human beta cells but does not identify the less sulfated HS in alpha cells.</p>
<p> </p>
<p><u>Reference</u>:</p>
<p>Theodoraki A, Hu Y, Poopalasundaram S et al (2015) Mol Cell Endocrinol 399: 296-310.</p>

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