Moflo machine

The flow cytometric data were collected on a BD (Becton Dickinson, Franklin Lakes, NJ, USA) Calibur or a LSRII flow cytometer and analyzed using FlowJo software (TreeStar, Ashland, OR, USA) or Summit software (Beckman Coulter, Fullerton, CA, USA). All antibodies were purchased from BD PharMingen or eBiosciences (CA, USA) as folollows: PE- or BV786- conjugated c-Kit, PE-Cy7-conjugated CD11b, PerCP-Cy5.5-conjugated Gr-1, APC-conjugated CD11c, APC-conjugated Annexin V and APC-conjugated Ki67. Total cells were Fc-blocked and stained with indicated combinations of antibodies for 30 min on ice, then washed three times and resuspended in 1% FBS/PBS. For apoptosis analysis, cells were resuspended with binding buffer and stained with Annexin V and 7AAD for 15 min at RT (25 °C) in the dark. For cell cycle analysis, cells were thoroughly resuspended and incubated with fixation and permeabilization solution for 20 min at room temperature, washed twice with BD Perm/Wash buffer and stained with HO33342 for 10 min. For cell sorting, the nucleated cells were stained with the indicated antibodies and resuspended in 2% FBS/PBS. The cells were sorted using a MoFlo machine (Beckman Coulter).

Kidneys were removed from both the control and UDC treated groups of mice and assessed to determine the presence of DsRed + cells using fluorescence-activated cell sorting (FACS). Kidney tissue was finely minced with surgical scalpels and subsequently digested with a solution of Hank’s Balanced Salt Solution (Life Technologies, Inc, Invitrogen, Carlsbad, CA) containing HEPES (25 mM), collagenase B (1 mg/ml, Roche Diagnostics, Indianapolis, IN), and deoxyribonuclease (0.1 mg/ml, Sigma-Aldrich, St. Louis, MO) for 60–90 min at 37 °C. All samples were filtered using 70-μm cell strainers and centrifuged at 3000 rpm at 4 °C for 5 min. Cell pellets were suspended in FACS buffer and then sorted via FACS on a Beckman Coulter MoFlo machine (Beckman Coulter, San Jose, CA). Acquired data were analyzed using the FlowJo V10 software (Tree Star, Ashland, OR) for three independent experiments.

Lung, spleen, liver, and brain tissue were assessed to determine the presence of DsRed⁺cells using FACS. The specimens were finely minced with surgical scalpels, and subsequently digested with a solution of Hank’s Balanced Salt Solution (Life Technologies, Inc, Invitrogen, Carlsbad, CA, USA) containing HEPES (25 mM), collagenase B (1 mg/ml, Roche Diagnostics, Indianapolis, IN, USA), and deoxyribonuclease (0.1 mg/ml, Sigma-Aldrich, St. Louis, MO, USA) for 60–90 min at 37°C. All samples were filtered using 70-μm cell strainers and centrifuged at 3,000 rpm at 4°C for 5 minutes. Cell pellets were suspended in FACS buffer and then sorted via fluorescence activated cell sorting (FACS) on a Beckman Coulter MoFlo machine (Beckman Coulter, San Jose, CA, USA). Acquired data were analyzed using the FlowJo V10 software (Tree Star, Ashland, OR, USA) for three independent experiments.