C2139

We cut the colons of euthanised mice into small pieces, which were serially incubated in 1 × HBSS containing 5% foetal bovine serum (FBS) and 2 mM EDTA, and Digestion Buffer containing 1 × HBSS supplemented with 5% FBS, collagenase VIII (1.5 mg/ml; C2139, Sigma-Aldrich), and DNase I (40 μg/ml; DN25, Sigma-Aldrich). The released cells were subjected to density gradient separation with 40% and 80% Percoll solutions (GE Healthcare). The cells located between the upper and lower phases were collected and stained with APC-conjugated anti-CD45 (30-F11), PE-conjugated anti-CD11b (M1/70), PE/Cy7-conjugated anti-CD11c (N418), FITC-conjugated anti-CD64 (X54-5/7.1), PE/Dazzle™ 594-conjugated anti-Ly6C (HK1.4), and APC/Cy7-conjugated anti-MHC-II (M5/114.15.2). All antibodies were purchased from BioLegend. We used a Zombie UV Fixable Viability Kit (BioLegend) to determine cell viability. Flow cytometric analysis was performed on a MoFlo™ XDP (Beckman Coulter), and the data were analysed with FlowJo® ver. 10 (Tree Star). If necessary, the cells of interest were sorted. Flow cytometric analysis was performed in the Stem Cell Laboratory, Medical Research Institute, TMDU.

Kidneys were harvested under sterile conditions from P6 WT and Pkhd1 null mice, followed by mincing the kidneys into 1 mm³ cubes and incubating in collagenase II digestion (0.1 g/ml, Sigma C2139) solution at 37 °C with shaking for 30 mins. Tubule cells were collected by centrifugation and resuspended in DMEM/F12 containing DNase I (2U/ml, Sigma D4263). After 3 mins of DNase I digestion, tubule cells were collected by centrifugation and the resuspended pellet was filtered through 70 µm pore filters to isolate tubules and exclude glomeruli. The tubules were washed in ice-cold PBS and pulsed in a centrifuge three times to remove collagenase and red blood cells. After each spin, the pellets become increasingly opaque white. The remaining pellets are isolated kidney tubule fragments.

Adipose tissues were minced in PBS containing 0.075% collagenase (Sigma-Aldrich, C2139, Saint Louis, MO, USA). After being incubated at 37 °C for 30 min and filtrated with a 100-mesh filter, cell suspensions were centrifuged at 1500 rpm for 5 min to remove adipocytes. Isolated stromal vascular fraction (SVF) pellet was collected from the bottom. The SVF pellet was resuspended in PBS containing 3% BSA, then red blood cell lysis buffer was added and incubated for 3 min. After washing in 3% BSA, the bottom cells were incubated as described previously [38 (link)].

SCAT and VAT were weighed, washed twice in PBS 1X 5% fetal bovine serum (FBS), cut into pieces of 2 to 3 mm and then digested in a bath of collagenase (C2139, Sigma) at a concentration of 0.33 mg /mL in DMEM supplemented with 5% FBS for 30 min at 37°C with constant shaking. Mechanical dissociation by suction/discharge with a 10 mL syringe was then performed. Next, the adipose suspension was filtered through a 100 micron mesh. Following an initial low-speed centrifugation (300g, 10 min), adipocytes (the upper phase) were separated and the lower phase (comprising the SVF cells) was centrifuged further. After two additional washes, the pellet containing the SVF was resuspended in PBS with 5% FBS. SVF cell suspensions were then counted in Malassez counting chambers (C-chip, NanoEntek, Seoul, Korea) under the microscope, using Trypan blue to exclude dead cells. SVF was either directly frozen for molecular analyses, frozen in FBS 10% DMSO for cell preservation, and/or sorted or analyzed immediately by flow cytometry.

The BAT derived from C57BL/6 mice was finely chopped in balanced salt solution of Hanks with collagenase (C2139; Sigma) and bovine serum albumin (A9647; Sigma). Tissue samples were incubated with hot water at 37 °C for 30 min under constant shaking. The cell suspension was filtered through a 100-μm cell strainer and centrifuged at 500 × g for 5 min. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)- high glucose (D5796; Sigma) and immortalised by infection with SV40 lentivirus (Applied Biological Materials Inc.). Introduction of SV40 lentivirus into cells was confirmed from the expression of SV40 lentiviral gene with quantitative reverse transcription polymerase chain reaction (qRT-PCR) method. These cells were called as CB-1 cells.