Sep 27, 2023
KAPP-Sen TMC: Dissociation of Pancreatic Acinar and Ducts (non-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 Acinar and Ducts (non-recovered). protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyjnorlx9/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: 85795
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 acinar and ducts were dissociated as follows.
Cell Dissociation with TrypLE
Cell Dissociation with TrypLE
Note
Before beginning cell dissociation coat all the materials (pipettes, tubes, etc.) with stopping media to prevent sticking. Stopping Media: DMEM + 10% FBS + 1:100 Glutamate.
Distribute specimens into 50 ml conical tubes, try to make similar cell concentrations across all tubes. *** Receive acinar in ~300 ml, should distribute into 12 tubes with 25 ml each***
Centrifuge acinar samples for 2 min at 230 g and 4°C. Leave duct samples to settle without disturbance.
Wash samples 3X with 25 ml PBS.
Add 10 ml TrypLE E and incubate at 37°C, mixing with a pipette approximately every 5 min.
- Acinar: in TrypLE E at 37°C for approximately 30 mins.
- Ducts: in TrypLE E at 37°C for approximately 15 mins.
To stop reaction, add 30 ml per tube of stopping media.
Centrifuge acinar and ducts at 1300 rpm, 4°C, 2 mins.
To stop: add 30 ml per tube of stopping media.
Centrifuge acinar and ducts at 1300 rpm, 4C, 2 mins.
Aspirate TrypLE E and wash 1X with stop media. Aspirate then resuspend in stop media to (Acinar) 5 ml or (Ducts) 2 ml.
Transfer cell suspension through 70 µm filter then combine to 2 ml (Acinar) and 1ml (Ducts). Filter again 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