Aug 26, 2025
Preparation of single-cell suspension from mouse pancreas
- hanjingnk 1
- 1Tsinghua university
External link: https://doi.org/10.1038/s43018-025-01065-3
Protocol Citation: hanjingnk 2025. Preparation of single-cell suspension from mouse pancreas. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7n18klwz/v1
Manuscript citation:
Han, J., Lu, X., Guo, M. et al. Spatiotemporal control of SMARCA5 by a MAPK–RUNX1 axis distinguishes mutant KRAS-driven pancreatic malignancy from tissue regeneration. Nat Cancer (2025). https://doi.org/10.1038/s43018-025-01065-3
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 24, 2025
Last Modified: August 26, 2025
Protocol Integer ID: 225307
Keywords: cell suspension from mouse pancreas preparation, mouse pancreas preparation, cell suspension
Abstract
Preparation of single-cell suspension from mouse pancreas
Guidelines
1. Finely dissect the pancreas into small fragments using a blade.
2. Subject fragments to digestion in a solution containing 1 mg/ml collagenase type V (Solarbio, C8170) in DMEM/F12 (Gibco, C11330500BT) for 5-10 minutes at 37 °C.
3. Halt the enzymatic reaction by diluting the collagenase solution with chilled (4 °C) PBS.
4. Obtain cell pellets through centrifugation at 300g for 5 minutes at 4 °C.
5. Further digest pellets with 1 ml TrypLE Express (Gibco, 12604013) at 37 °C.
6. Terminate the reaction by diluting the solution with chilled (4 °C) PBS, and collect cell pellets by centrifugation at 300g for 5 minutes at 4 °C.
7. Incubate the cell pellet with a 200 U/ml DNase I (Solarbio, D8070) solution in DMEM/F12. Terminate the reaction by diluting with chilled (4 °C) PBS, and collect cell pellets by centrifugation at 300g for 5 minutes at 4 °C.
8. Resuspend the cells in chilled sorting buffer (PBS pH 7.2 containing 0.5% (wt/vol) BSA and 2 mM EDTA) for subsequent FACS analysis.
9. For mice treated with caerulein, further stain the cells with APC-conjugated CD45 antibody to exclude immune cells.
Materials
Pancreas tissue; blade (for dissection); Collagenase type V (Solarbio, C8170) prepared at 1 mg/ml in DMEM/F12 (Gibco, C11330500BT); DMEM/F12 (Gibco, C11330500BT); TrypLE Express (Gibco, 12604013); DNase I (Solarbio, D8070) prepared at 200 U/ml in DMEM/F12; Phosphate-buffered saline (PBS), chilled (4 °C); Sorting buffer: PBS pH 7.2 containing 0.5% (wt/vol) BSA and 2 mM EDTA; BSA; EDTA; APC-conjugated CD45 antibody (for exclusion of immune cells); Centrifuge capable of 300g; Equipment for FACS analysis.
Troubleshooting
Safety warnings
This protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Ethics statement
This protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Before start
Prepare chilled (4 °C) PBS. Prepare collagenase type V at 1 mg/ml in DMEM/F12. Prepare DNase I at 200 U/ml in DMEM/F12. Prepare sorting buffer: PBS pH 7.2 with 0.5% (wt/vol) BSA and 2 mM EDTA. Pre-chill centrifuge and keep reagents and tubes on ice or at 4 °C where indicated. Have APC-conjugated CD45 antibody available if excluding immune cells (e.g., in caerulein-treated mice).
Finely dissect the pancreas into small fragments using a blade.
Subject fragments to digestion in a solution containing 1 mg/ml collagenase type V (Solarbio, C8170) in DMEM/F12 (Gibco, C11330500BT) for 5–10 minutes at 37 °C.
Halt the enzymatic reaction by diluting the collagenase solution with chilled (4 °C) PBS.
Obtain cell pellets by centrifugation at 300g for 5 minutes at 4 °C.
Further digest the pellets with 1 ml TrypLE Express (Gibco, 12604013) at 37 °C.
Terminate the TrypLE reaction by diluting the solution with chilled (4 °C) PBS and collect cell pellets by centrifugation at 300g for 5 minutes at 4 °C.
Incubate the cell pellet with 200 U/ml DNase I (Solarbio, D8070) in DMEM/F12; terminate the reaction by diluting with chilled (4 °C) PBS and collect cell pellets by centrifugation at 300g for 5 minutes at 4 °C.
Resuspend the cells in chilled sorting buffer (PBS pH 7.2 containing 0.5% (wt/vol) BSA and 2 mM EDTA) for subsequent FACS analysis.
For mice treated with caerulein, further stain the cells with APC-conjugated CD45 antibody to exclude immune cells.