Embryonic Kidney Dissociation to single cells (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Thomas Carroll; Alicia Fessler

Keywords

[‘Cell dissociation’, ‘kidney’, ‘kidney digestion’, ‘mouse’, ‘tissue dissociation’]

Subjects

[‘Cell biology’, ‘Isolation, purification and separation’]

Release Date

2020-08-20

Abstract

Embryonic Kidney Dissociation to single cells for FACS enrich for interstitial cells

Reagents

Collagenase A (Sigma, 101378001) Pancreatin (porcine) (Sigma, P1750) PBS without calcium or magnesium HBSS without calcium or magnesium (Life Technologies, 14175-095) FBS AutoMACS running buffer (Miltenyi, 130-091-221)

Equipment

Razorblades 30 um pre-separation filter (Milteni Biotec, Cat. 130-041-407) cell-strainer cap (Falcon, Cat. 352235) 5mL Polypropylene round bottom tube (Globe Scientific, Cat. 110428) Benchtop centrifuge Transfer pipette Nutator placed at 37C

Procedure

**NOTE: this protocol is for enrichment of interstitial cells via FACS. However, capsule is removed, most glomeruli are filtered and the ureter is removed prior to dissociation. Thus, capsular, mesangial cells and urethelial interstitium will not be enriched using this protocol.

Prepare enzyme digest prior to isolation

Prepare collagenase A/pancreatin enzyme digest solution 2 hours before kidney dissection by adding 25 mg collagenase A to 10 ml PBS without calcium or magnesium. Place on a nutator at RT for 15 minutes or until collagenase A is dissolved. Add 100 mg pancreatin and place on nutator until kidney harvest is complete. Filter sterilize (0.2 μm filter) before use to remove any undissolved particles. Alternatively, large batches of the enzyme digest solution can be made, filter sterilized, and frozen at -20°C for up to 6 months. Protocol from Brown et al. 2015 or from [[N-H9AM]]

Dissociation of mouse kidney into single cells for FACS

1. Dissect the kidneys from E13 to P1 Foxd1Cre;R26R-Tomato mice in a 10 cm dish containing a sufficient volume of cold PBS without calcium or magnesium to cover the embryos. Completely remove the ureter and kidney capsule and transfer kidneys to the HBSS-containing tube using a transfer pipette with an orifice cut to at least twice the diameter of the kidneys to be transferred.
2. Wash kidneys in HBSS for 2 minutes at 37C on a nutator
3. Mince kidneys using two razorblades on ice
4. Digest no more than 4 pairs of kidneys in 2mL enzyme digest for 8 minutes at 37C on a nutator using manual dissociation via pipetting through a P1000 tip every 2 minutes.
5. Inactivate digestion by adding 125uL FBS.
6. Pellet the cells at 400g for 5 minutes
7. Discard supernatant, and resuspend cells in 1mL autoMACS running buffer
8. Place a 30 μm pre-separation filter on a 15 ml conical tube and wash with 4 ml of autoMACS running buffer. Discard the flow through.
9. Add cell suspensions to the washed 30 μm pre-separation filter and wash with 500 μl of autoMACS running buffer
10. Pellet the cells at 400g for 5 minutes
11. Discard supernatant, and resuspend cells in 0.5mL autoMACS running buffer
12. Pass cell suspension through a cell-strainer cap attached to a 5mL polypropylene round bottom tube at least two times
13. Resuspend cells to a concentration of 100,000-1,000,000 cells/mL prior to submitting for FACS

Consortium

(Re)Building a Kidney (RBK) Consortium