Jan 25, 2019
Version 1
Sampling of Human Islets for Quality Control Purposes V.1
- 1University of Alberta
- Human Cell Atlas Method Development Community
- CIRTNR2FIC
External link: https://doi.org/10.1210/endocr/bqae079
Protocol Citation: James Lyon, Aliya F Spigelman, Jocelyn E Manning Fox, Patrick E Macdonald 2019. Sampling of Human Islets for Quality Control Purposes. protocols.io https://dx.doi.org/10.17504/protocols.io.sizecf6
Manuscript citation:
Banerjee R, Zhu Y, Brownrigg GP, Moravcova R, Rogalski JC, Foster LJ, Johnson JD, Kolic J (2024) Beta-Hydroxybutyrate Promotes Basal Insulin Secretion While Decreasing Glucagon Secretion in Mouse and Human Islets. Endocrinology 165(8). doi: 10.1210/endocr/bqae079
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 10, 2018
Last Modified: January 25, 2019
Protocol Integer ID: 14649
Materials
MATERIALS
Sodium Citrate Dihydrate Fisher ScientificCatalog #S279
Sodium Chloride Fisher ScientificCatalog #BP358
Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) Merck MilliporeSigma (Sigma-Aldrich)Catalog #ED2SS
Phosphate Buffered Saline (PBS)Fisher ScientificCatalog #BP24384
Z-fixFisher ScientificCatalog #NC9378601
ART® 1000G Self-Sealing Barrier Pipet Tips Molecular BioProductsVWR International (Avantor)Catalog #53509-504
ART® 200G Self-Sealing Barrier Pipet Tips Molecular BioProductsVWR International (Avantor)Catalog #53509-502
95% Ethyl AlcoholFisher ScientificCatalog #HC13001GL
Acetic Acid Glacial ACS Grade ≥ 99.7% Fisher ScientificCatalog #LC101003
Hydrochloric acid - concentratedFisher ScientificCatalog #A149C4
Islet Suspension
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 preparation
Using a 25ml pipette and pipette aid, completely suspend the islet preparation.
Sampling - Insulin and DNA samples
Sampling - Insulin and DNA samples
Using a pipette with a wide bore ART® 200G Self-Sealing Barrier Pipet Tip, remove 4x 50µl samples from the islet suspension and transfer each sample to a 5ml polypropylene tube for DNA and insulin samples. See steps 5 & 6.
Sampling - Islet Equivalent counts
Sampling - Islet Equivalent counts
Using a pipette with a wide bore ART® 200G Self-Sealing Barrier Pipet Tip, remove one 50µl sample from the 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.)
Sampling - Islet for Immunohistochemistery
Sampling - Islet for Immunohistochemistery
Using a pipette with an ART® 1000G Self-Sealing Barrier Pipet Tip, remove one 500µl sample from the islet suspension and transfer to a 5ml polypropylene tube for histology. See step 7.
DNA samples
DNA samples
Add 4.5ml citrate buffer to each of 2 of the above samples step 2.
Centrifuge at 1500 rpm for 5 min.
00:05:00
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 4•C until dsDNA assay.
Citrate Buffer - 1 Litre
To 800ml 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 3.15 g
- Sodium Chloride 8.77 g
- Disodium EDTA 1.01 g
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 –20•C. Thaw the buffer as needed and store at 4oC.
Insulin samples
Insulin samples
Add 950µl of Acid/EtOH solution to each of the remaining two samples (step 2).
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 -20•C to await insulin assay.
Acid/Ethanol solution
Add the following reagents in a sealed glass bottle. Store at 4•C prior to use.
- 150ml 95% ethanol
- 47ml acetic acid
- 3ml concentrated hydrochloric acid
Samples for histology
Samples for histology
Add 4.5ml PBS to the 500µl sample (step 4) and allow the suspension to settle to a pellet for at least 5 minutes.
Remove the supernatant and add 500µl of Z-fix and transfer to a 1.5ml Eppendorf tube
Store tube @4°C overnight.
Remove the fixative and add 500µl PBS and store @4°C to await processing for histology
Submit to histology core for processing to blocks and slides.
Once the processed tissue is returned from histology store all blocks and slides in the histology library and record inventory on REDCap.
Library for Histology
Library for Histology
Verify and record the sample type and location in the Histology Library record.
Sample Histology Library record:
| Isolation Number | Sample | ||||||||||
| Pancreas Biopsy | Post-isolation | Pre-distribution | Pre-cryopreservation | Post-cryopreservation | |||||||
| Blocks | Slides | Blocks | slides | Blocks | slides | Blocks | slides | Blocks | slides | ||
| R001 | 1 | 4 | 1 | 4 | 1 | 4 | 1 | 4 | 1 | 4 | |
| R002 | 1 | 4 | 1 | 4 | 1 | 4 | 1 | 4 | 1 | 4 | |
| R003 | 1 | 4 | 1 | 4 | 1 | 4 | 1 | 4 | 1 | 4 | |
| R004 | 1 | 4 | 1 | 4 | 1 | 4 | 1 | 4 | 1 | 4 | |
| R005 | 1 | 4 | 1 | 4 | 1 | 4 | 1 | 4 | 1 | 4 | |
| R006 | 1 | 4 | 1 | 4 | 1 | 4 | 1 | 4 | 1 | 4 | |
| R007 | 1 | 4 | 1 | 4 | 1 | 4 | 1 | 4 | 1 | 4 |