Kim1-GCE;tdTom Cell isolation protocol (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; Monica Chang-Panesso

Keywords

[‘genetic labeling’, ‘kidney injury’, ‘proximal tubule epithelial cells’]

Subjects

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

Release Date

2016-10-21

Abstract

This protocol allows isolation of tdTomato-positive dedifferentiated mouse proximal tubule cells from postischemic kidneys of Kim-1-GFPCreERt2; R26tdTomato bigenic mice. Tamoxifen is administered 6 hours prior to IRI and at 24 hours after IRI, with kidney isolation at 72 hours post-IRI.

Reagents

• Liberase TL Research grade. Roche, Cat# 05401020001
• DNaseI. Roche, Cat# 10104159001
• DMEM/F12. Gibco, 11330-032
• 1x PBS. Gibco, 10010023
• Accutase. Innovative Cell Technologies, Cat# AT104
• 70 л_M strainer
• 40 л_M strainer
• Disposable scalpel No.10. McKesson, Cat 16-63110.

Culture Media: To prepare 250 mL media:

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Equipment

• GentleMACS Dissociator. Miltenyi Biotec, Cat #:130-093-235.
• C-tubes. Miltenyi Biotec, Cat# 130-093-237.

Procedure

Solutions:

Liberase enzyme solution:

• Dissolve 5 mg vial in 1mL of RNAse free water
• Make 250 л_L aliquots, store at -20C

DNaseI solution:

• Dissolve 10 mg DNaseI in 1mL PBS
• Make 50 л_L aliquots, store at -20C

Liberase working solution:

• Take 5mL of DMEM/F12
• Add 250 л_L Liberase enzyme and 50 л_L DNaseI solution
• Filter sterilize with 0.22 л_M filter

Cell Isolation protocol

1. Anesthesize mice with isoflurane.
2. Perfuse heart with sterile PBS.
3. Remove kidneys and place in petri dish with PBS. From now on, work in tissue culture hood whenever processing tissue.
4. Remove kidney capsule using sterile forceps.
5. Using a sterile scalpel, cut kidney in half (sagittal plane). One half from each kidney will be used for cell isolation. The remaining halves can be saved for RNA, protein, OCT and paraffin.
6. Mince kidney using sterile scalpel.
7. Put minced kidney in C-tube (for MACS dissociator)
8. Add 2 mL Liberase working solution to C-tube per kidney.
9. Run D-01 program on MACS dissociator
10. Shake for 10 min at 37C
11. Run D-01 program on MACS dissociator
12. Filter through 70 л_M strainer on 50-mL Falcon tube
13. Add 4 mL of DMEM/F12 with 10% FBS
14. Spin 5 min at 500 rcf
15. Wash the cells with PBS
16. Spin 5 min at 500 rcf
17. Resuspend the pellet in 2 mL Accutase and transfer to C-tube again.
18. Shake for 10 min at 37C
19. Run D-01 program on MACS dissociator
20. Add 4 mL of DMEM/F12
21. Filter through 40 л_M on 50-mL Falcon tube.
22. Spin 5 min at 500 rcf
23. Plate the pellet by resuspending in final culture media.

Cell Culturing

1. Media is removed 48hrs after initial plating
2. Cells are rinsed with 1X PBS
3. Fresh media is added

FACS sorting (preliminary protocol, need to be optimized)

1. Remove culture media
2. Rinse cells with 1X PBS
3. Detach cells with Accutase as per manufacturer protocol.
4. Collect cells and centrifuge at 500 rcf x 4 min
5. Aspirate supernatant and resuspend pellet in FACS buffer (1X PBX + 1% FBS)
6. Transfer to FACS sorting tube (Falcon, 352235)
7. Proceed to FACS sorting.

Timing

~1-2 hrs

Anticipated_Results

• In about 3 days, there will be a small groups of Kim1-GCE/tdTom cells.

References

Ichimura et al. Kidney injury molecule-1 is a phosphatidylserine receptor that confers a phagocytic phenotype on epithelial cells. J Clin Invest.118, 1657-68 (2008).

Consortium

(Re)Building a Kidney (RBK) Consortium