TITLE: Sampling of Human Islets for Quality Control Purposes
AUTHORS: James Lyon, Aliya Spigelman, Jocelyn E Manning Fox, Patrick Macdonald

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol described the sampling of human islets for quality control purposes, as performed by the Alberta Diabetes Institute IsletCore. </span><a href="http://www.bcell.org/adi-isletcore.html" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">http://www.bcell.org/adi-isletcore.html</span></a></div></div>

[STEPS]
?. [Solution Preparation]
To  of Milli-Q water add the following reagents and allow to mix into solution. The citric acid will not completely go into solution until the pH is set to 7.4   Sodium Citrate Dihydrate               Sodium Chloride     Disodium EDTA        Bring to volume with Milli-Q water and set the pH to 7.4.Aliquot the Citrate buffer into 50ml conical tubes and store at . Thaw the buffer as needed and store at .
800 mL
3.15 g
8.77 g
1.01 g
-20 °C
4 °C
?. [Islet Suspension]
Suspend the islet preparation in a known volume of culture media (typically 50ml x2 for 100ml total volume). See Human Islet Isolation Media Preparation for CMRL preparationUsing a 25ml pipette and pipette aid, completely suspend the islet preparation by gently pipetting up and down.
?. [Sampling - Islet Equivalent counts]
Using a pipette with a wide bore ART® 200G Self-Sealing Barrier Pipet Tip, remove one  sample from the centre of islet suspension and transfer the sample to a petri dish for an islet equivalent count performed twice on the same sample (Refer to Human Islet Quantification and Purity Assessment protocol.)
50 µl
?. [Sampling - DNA and insulin samples]
Using a pipette with a wide bore ART® 200G Self-Sealing Barrier Pipet Tip, remove 4x  samples from the centre of the islet suspension and transfer each sample to a 5ml polypropylene tube for DNA (x2) and insulin samples (x2).
50 µl
?. [DNA samples]
Add  citrate buffer to each of the 2 samples indicated for DNA in step 5.Centrifuge at  .
4.5 mL
Centrifuge: 1500 33, 5 min
?. [Insulin samples]
Add  of Acid/Ethanol solution to each of 2 of the above samples (step 5).Label these sample tubes with the internal identifier number (Rxxx), sample number (a or b), and sample date.Cap and store the sample tubes at  to await insulin assay.
950 µl
-20 °C
?. [Histology Samples]
Using a pipette with an ART® 1000G Self-Sealing Barrier Pipet Tip, remove one  sample from the islet suspension and transfer to a 5ml polypropylene tube labelled with the internal identifier number (Rxxx) for histology. Add  PBS to the  sample and allow the suspension to settle to a pellet for at least .Remove the supernatant and add  of Z-fix and transfer to a 1.5ml Eppendorf tubeStore tube at .Remove the fixative and add  PBS and store at   to await processing for histologyEmbed fixed islet sample in low melt agarose and submit to histology core for processing to paraffin-embedded blocks and sections.Once the processed tissue is returned from histology store all blocks and slides in the histology library.
500 µl
4.5 mL
500 µl
500 µl
4 °C
500 µl
4 °C
?. [Solution Preparation]
Acid/Ethanol solutionAdd the following reagents in a sealed glass bottle.  Store at  prior to use.   95% ethanol      acetic acid        concentrated hydrochloric acid
4 °C
150 mL
47 mL
3 mL
?. [DNA samples]
Using an aspirating pipette and a pipette tip remove all the resulting supernatant from the pelleted islets.Label the sample tube with the internal identifier number (Rxxx), sample number (1 or 2), and sample date. Cap and store the sample tubes at  until dsDNA assay.
4 °C