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Isolation of single cells from murine late distal convoluted tubules and connecting tubules
Author(s) -
Jensen Thomas Buus,
Jensen Uffe Birk,
Fenton Robert A,
Praetorius Helle A,
Praetorius Jeppe
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.863.7
Subject(s) - collagenase , propidium iodide , green fluorescent protein , chemistry , microbiology and biotechnology , distal convoluted tubule , cell culture , biophysics , kidney , reabsorption , biology , biochemistry , apoptosis , endocrinology , enzyme , genetics , programmed cell death , gene
Proteomic studies of late distal convoluted tubule (DCT2) and connecting tubule (CNT) cells have so far been hindered by technical difficulties in isolating these cells with sufficient yield and purity. We exploited a mouse model with endogenous expression of enhanced green fluorescent protein (EGFP) driven by the transient receptor potential vallinoid 5 (TRPV5) channel promoter. Whole kidneys were minced and incubated with 1 mg/ml proteinase K and 1 mg/ml collagenase II for 45 min at 37°C agitated at 180 RPM. The tubule‐suspension was treated with 0.12% trypsin in Ca 2+ ‐free buffer for 2 × 5 min to obtain single cells. Cell separation and morphology were evaluated by light microscopy. Blood vessels and cell clusters were removed by passing the cell suspension through a 40‐μm mesh before fluorescence activated cell sorting. Cells were sorted on a FACS AriaIII (BD) with 100 μm nozzle at 20 psi and 488 nm excitation. Dead cells (approximately 2%) were excluded using propidium iodide. By this method, the yield corresponded to approximately 2.5 × 10 7 cells per mouse kidney of which 6×10 5 were GFP positive (2.4% of all cells) or 38 μg protein per kidney. Thus, we have developed a protocol that may allow proteomic analysis of mouse DCT2/CNT cells with virtually no contamination with cells from other renal structures or with intercalated cells from DCT2/CNT. Supported by the Danish Council for Independent Research | Medical Sciences.