Digestion protocol for kidney single cell suspension (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
Racheal Jappert; Jeffrey Pippin; Ying Zheng; Stuart Shankland
Keywords
[‘kidney digestion’, ‘FACS’, ‘MACS cell separation’]
Subjects
[‘Isolation, purification and separation’, ‘Cell culture’]
Release Date
2017-05-25
Abstract
Describes the procedure for digesting mouse or human kidney into single cell suspensions for isolation by FACS or MACs kits.
Reagents
Liberase
- Sigma 5401020001 Liberase™ TL Research Grade low Thermolysin concentration
- Prepare Liberase in sterile LPS free water: (10µg/µl)
- Leave on ice for 30 min.
- Sterile filter through 0.22µm syringe filter
- Make 100µl aliquots and freeze
- (Stock Stored in Liberase/DNAse Enzyme Box, Shankland -20oC Freezer E146)
DNAse
- Sigma 4716728001 DNase I recombinant, RNase-free
- Prepare DNAse (10units/µl) in serum free media
- Make 50µl aliquots and freeze
- (Stock Stored in Liberase/DNAse Enzyme Box, Shankland -20oC Freezer E146)
Liberase/DNAse Mix
- Add to 5ml media (no serum), 1 aliquot Liberase (100µl) and 1 aliquot (50µl) DNAse (good for 2 kidneys)
Ammonium chloride RBC lysis Buffer
- (10X concentration)
- NH4Cl (ammonium chloride) 8.02gm
- NaHCO3 (sodium bicarbonate) 0.84gm
- EDTA (disodium) 0.37gm
- QS to 100ml with sterile Millipore water. Store at 4°C for six months.
- Working solution
- Dilute 10ml 10X concentrate with 90 ml sterile Millipore water. Refrigerate until use.
FACS Buffer
- Use culture media for sorting, phenol red free and low serum: Podocyte culture media is ideal for sorting and growth media for the cells being isolated works well for collecting cells
Cell strainers
- Fisher 08-771-19 100µm
- Fisher 08-771-1 40µm
Procedure
- Perform cardiac puncture and flush kidneys with sterile cold PBS, (isolation can be performed without PBS perfusion, but recommend performing RBC lysis step below)
- Transfer kidneys into 50ml conical tube
- In biosafety cabinet, wash kidneys 5 times with sterile saline
- Dissect and de-capsulate kidneys
- Mince kidneys in sterile 10cm dish with sterile scalpel or sterile razor blade until finely minced
- Transfer minced kidney into Liberase/DNAse Mixture (2-2.5ml/kidney) in 15ml tubes
- Agitate vigorously in shaking water bath 37oC for 30 min, vortex every 10min
- Mechanically disperse cell clumps by passing the digested kidney through a 22G needle 5-10 times
- Add 2.5ml/kidney of media containing FCS (this neutralizes the digestion)
- Pass first through a 100µm pore size cell strainer
- Then pass through a 40µm pore size cell strainer
- Spin at 4°C at 250 x g for 5 minutes
- (Optional)Fill 15ml tube to capacity with fresh cold 1x RBC lysing solution (see above)
- (Optional) Invert or rock for ~10 minutes at room temperature until liquid is clear red
- (Optional) Spin at 4°C at 250 x g for 5 minutes
- Re-suspend in FACS buffer (see above), 1-2ml/kidney
- Proceed to FACS facility, make sure to have collection tubes
Consortium
(Re)Building a Kidney (RBK) Consortium
