Sep 27, 2023
KAPP-Sen TMC: Dissociation of Pancreatic Islets (recovered)
- Juliana Alcoforado Diniz1,
- Jessica Garofalo1,
- Dylan Baker1,
- Paul Robson1,2,3
- 1The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA;
- 2Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, Farmington, CT, USA;
- 3Institute for Systems Genomics, University of Connecticut, Farmington, CT, USA
Protocol Citation: Juliana Alcoforado Diniz, Jessica Garofalo, Dylan Baker, Paul Robson 2023. KAPP-Sen TMC: Dissociation of Pancreatic Islets (recovered). protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9pyq4g3e/v1
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 01, 2023
Last Modified: September 27, 2023
Protocol Integer ID: 85794
Keywords: SenNet KAPP-Sen TMC
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Abstract
The dispersed samples were shipped cold from PRODOLABS. Prior to scRNA-seq dispersed samples from brain dead donor’s pancreatic islets were recovered and dissociated as follows.
Cell Dissociation with Accutase
Cell Dissociation with Accutase
Note
Before beginning cell dissociation coat all the materials (pipettes, tubes, etc.) with PIM-S001GMP media to prevent sticking.
Transfer cell suspension of pure islets to a new 50ml tube. Use additional media to rinse original container.
Centrifuge at room temperature 130g for 5 mins.
Aspirate the supernatant and add media to the appropriate concentration of 1,000 islets/1 ml.
--> Ex: If receiving 7,000 islets, use 7 ml media.
To recover cells, add half of the islets to a coated flask and incubate at 37°C overnight.
Coat pipette and add islets from flask to a 50ml tube. Wash flask with media to make sure all islets have been collected.
Centrifuge at 130 g, room temperature for 2 mins.
Aspirate media and resuspend in 4 ml accutase. Incubate at 37C for 8 mins, mixing with pipette every 2 mins. Check at 6 mins. -->1 mL accutase/1,000 islets
Add CMRL 1066 (Cat. 11530037) media to 25 ml then centrifuge at 230 g.
Aspirate supernatant and resuspend in 1.5 ml of CMRL
Filter through a 40 µm Flowmi.
Count cells using AO/PI (acridine orage/propidium iodide) Cell Viability Kit for Luna-FL automated cell counter.
Proceed to cell fixation.
Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling
Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling
Cells were fixated prior to scRNAseq according to https://dx.doi.org/10.17504/protocols.io.x54v9py5zg3e/v1