TITLE: Quantitative Assessment of Islet Viability upon Arrival to HIPP by Staining with Trypan Blue Dye
AUTHORS: IIDP-HIPP 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;font-style:italic;">This Standard Operating Procedure (SOP) is based on the Vanderbilt Human Islet Phenotyping Program (HIPP) procedures for dispersing islets to single cell suspension and quantitatively assessing viability by Trypan Blue staining.</span></div><div class = "text-block">This SOP defines the assay method used by the Human Islet Phenotyping Program (HIPP) for quantitative and qualitative determination of the Purified Human Pancreatic Islet product, post-shipment, manufactured for use in the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)-sponsored research in the Integrated Islet Distribution Program (IIDP).</div></div>

[STEPS]
?. [Procedures]
Dispersion of islets into single cell suspension
?. [Procedures]
Prepare 2 aliquots of approximately 100 handpicked islets in tubes to determine viability of islets. Note: This is important especially for islet preparations with lower purity where cell death in the contaminating exocrine compartment may occur at higher rate compared to islets and thus could lead to underestimating islet viability.
1.5 mL
?. [Procedures]
Wash islets with 2mM EDTA made in 1X PBS and centrifuge them at 200 rcf for 1 minute at. Repeat two times.
4 °C
?. [Procedures]
After last wash, resuspend the pellet in  of Accutase.
500 µl
?. [Procedures]
Triturate islets at slow speed for 10 minutes at  using an electronic multichannel pipet with   tips. Note: As islets are getting dispersed, the sample should become slightly opaque.
37 °C
200 µl
?. [Procedures]
Quench the reaction with of CMRL-1066 media containing 10% FBS, 2mM L-glutamine, 1% penicillin/streptomycin.
500 µl
?. [Procedures]
Rinse the cells with  of CMRL-1066 medium, centrifuge at 500 rcf for 3 minutes at . Carefully remove the media without disturbing the pellet at the bottom of the tube. Repeat one more time.
1 mL
4 °C
?. [Procedures]
Resuspend the cell pellet in 50-75 µL of CMRL-1066 medium by gently flicking the tube and keep the sample on ice. The volume of CMRL used is determined by the relative size of the cell pellet.
?. [Procedures]
Cell counting
?. [Procedures]
From each islet sample, transfer two  aliquots of the cell suspension into  tubes. Mix well before pipetting the sample.
10 µl
1.5 mL
?. [Procedures]
Prepare the cell counting chamber slide for sample loading (slide has two chambers and looks similar to the hemocytometer but without grids).
?. [Procedures]
Centrifuge Trypan Blue stock at 3,000 rcf for 1 minute to pellet particulates that may be present in the dye.
?. [Procedures]
Gently mix the first aliquot of the islet cell suspension with of Trypan Blue dye and then load of the mixture into the chamber slide.
10 µl
10 µl
?. [Procedures]
Wait approximately 20 seconds for the cells to spread out on the slide, insert the slide into the Countess II cell counter and acquire cell counts following instrument guidelines. The output of cell and viability counts for the cell suspension of purified islets is illustrated in Figure 1.
?. [Procedures]
Repeat cell counts for the second aliquot of the same islet sample using the second slide chamber.
?. [Procedures]
Repeat steps 2.2 through 2.5 for the remaining sample.
?. [Procedures]
Note: Cells need to be imaged within 15 minutes of Trypan Blue addition due to cytotoxicity of the dye.
?. [Procedures]
Report average values of viable cells for duplicates of purified islets based on two cell counts per each replicate.
?. [Data Storage and Reporting]
Offload the data, including cell images, from the cell counter using a USB drive and transfer to the Vanderbilt HIPP server-based platform for storage and analysis.
?. [Data Storage and Reporting]
Islet viability data reporting will be uploaded within 1 business day to the IIDP-HIPP database and immediately disseminated to IIDP-affiliated investigators and islet isolation centers.
?. [Deviations and Resolutions]
Document any deviations that occurred during this protocol that affect the final results and report with the analysis of the assay.