Isoflow solution

To separate and collect GFP-positive (GFP+) and GFP-negative (GFP-) fractions, the cell suspension was passed through a MoFlo ^TM XDP fluorescence activated cell sorter (FACS) (Beckam Coulter) equipped with a 488 nm air-cooled argon solid state laser and a standard filter setup. An IsoFlow™ solution (Beckman Coulter) was used as the sheath fluid; the pressure of the sheath fluid was 60 psi, and the nozzle size was 70 μm. To minimize RNA degradation and keep cells alive, the sorted cells were collected directly into sterile 5-mL polypropylene tubes with Dulbecco′s Modified Eagle′s Medium (DMEM) (Merck). Moreover, after acquiring 100,000 events the tube was transferred on ice and replaced by a new one. Flow rate and the concentration of samples were adjusted to keep the acquisition lower than 500 events/s. The sorting procedure was stopped after acquiring 500,000 GFP+ cells. The settings used for the sorting were determined empirically and are provided as supporting information (S1 File). Immediately after the sorting procedure we isolated RNA from both GFP+ cells as well as unsorted material derived from the kidney tissue.

The phenotypic characteristics of PBMCs were analyzed by staining surface antigens with specific antibodies (Beckman Coulter, Solna, Sweden). Briefly, 50 µL of cell suspension was mixed with 2 µL of antibody solution and incubated for 15 min in the dark. The antibodies were directly conjugated to following fluorochromes: CD14-FITC, CD19-PC5.5, CD16-PC7, CD56-PC7, CD2-APC-AF750 and CD45-Krome orange in 250 µL optilyse C lysis solution (Beckman Coulter) and incubated for 15 min in the dark. To minimize autofluorescence, 250 µL of isoflow solution (Beckman Coulter) was added to the cell suspension and incubated for 15 min in dark. Analysis was performed by running the samples on a Navios Flow Cytometer (BD Biosciences, Stockholm, Sweden) and analyzed with Navios analysis software (BD Biosciences).