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Adult Mouse Kidney Dissociation (Version 1.0) | ATLAS-D2K Center

PLEASE NOTE: ATLAS-D2K closed July 31, 2025 and this website is for reference purposes only.

Adult Mouse Kidney Dissociation (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

Benjamin Humphreys

Keywords

[‘tissue dissociation’, ‘mouse’, ‘FACS’]

Subjects

[‘Experimental organisms’, ‘Isolation, purification and separation’]

Release Date

2017-05-04

Abstract

This protocol has been developed for dissociation of adult mouse kidney into a single cell suspension. We have verified that all major epithelial cell types are present in the dissociated cells, and the resulting suspension is suitable for use in either FACS analysis or single cell RNA sequencing applications. The presence of interstitial cell types has not yet been validated but this protocol will be updated when those results are available.

Reagents

Reagents for buffer and stock:

Materials Company Cat. No. Storage
10X HBSS Gibco 14065-056 RT
1M HEPES USB 16924 RT
PBS Gibco 10010-023 RT
Sodium Bicarbonate Sigma S6297-250g RT
Liberase TL Roche 05401020001 -20°C
DNase I Sigma 10104159001 -20°C
10x RBC lysis buffer BD 555899 4°C
DMEM/F12 Gibco 11320-033 4°C
0.5% Trypsin Gibco 15400-054 -20°C
0.4% Trypan Blue Gibco 15250061 RT

Buffers and Solutions

I. Stock solution Liberase enzyme stock:

  • Dissolve the 5 mg in the vial in 1 mL H2O
  • Put on ice 30 min.
  • Filter through 0.22 μm filter
  • Aliquot 250 μL, store at -20°C

DNaseI stock:

  • Weigh 10 mg (DNaseI from Bovine Pancreas, Sigma)
  • Dissolve in 1 mL PBS
  • Filter sterilize with 0.22 μm filter
  • Aliquot 50 μL per tube, store at -20°C

7.5% Sodium bicarbonate

  • Dissolve 0.75 g Sodium bicarbonate in 10 mL H2O

II. Working solution

Tissue collecting buffer:

  • 10 mL HBSS
  • 20 μL 1M HEPES buffer
  • 4.7 μL 7.5% Sodium bicarbonate

Liberase enzyme working buffer:

  • 5 mL DMEM/F12
  • add Liberase aliquot
  • add DNaseI aliquot

1x RBC lysis buffer

  • 100 μL RBC lysis buffer
  • 900 μL H2O

Equipment

Machines and tools:

Materials Company Cat. No.
gentleMACS Dissociator Miltenyi Biotec 130-093-235
Incu-Shaker BenchMark BMS:H1010
40 μm cell strainer VWR 89508-342

Procedure

  1. Anesthetize mice with isoflurane
  2. Perfuse heart with cold PBS
  3. Take out kidney and start processing, or store in ‘Tissue collecting buffer’ on ice until ready to continue
  4. Remove kidney capsule, and mince kidney into small pieces of 1-2mm
  5. Put minced kidney in C-tube (for gentleMACS dissociater)
  6. Add 2 mL Librase enzyme solution to C-tube per kidney
  7. Run D-01 program on gentleMACS dissociater*
  8. Incubate sample for 10 minutes at 37°C under continuous rotation *
  9. Run D-01 program on gentleMACS dissociater*
  10. Add 4 mL of DMEM/F12 with 10% FBS
  11. Spin 5 min at 800 rcf at 4°C
  12. Wash the cells with ice-cold PBS
  13. Spin 5 min at 800 rcf at 4°C
  14. Lyse the red blood cell with 1 mL RBC lysis buffer, incubate for 5 min at RT
  15. Spin 5 min at 800 rcf at 4°C
  16. Resuspend the pellet in 2 mL 0.5% Trypsin
  17. Incubate sample for 10 minutes at 37°C under continuous rotation *
  18. Run D-01 program on gentleMACS dissociater*
  19. Add 4 mL of DMEM/F12 with 10% FBS
  20. Filter through 40 μm cell strainer.
  21. Spin 5 min at 800 rcf at 4°C
  22. Wash with ice-cold PBS and pellet the cells at 800 rcf for 5min
  23. Resuspend the cells in ice-cold PBS and assess cell viability by Trypan blue staining
  • Longer incubation with enzyme needs less mechanical dissociation with gentleMACS. This will results in better cell viability, so one could play around with conditions here.

Consortium

(Re)Building a Kidney (RBK) Consortium