FACS staining

All antibodies were diluted in 5 ml of standard FACS buffer (PBS, 2% FSC and EDTA). After blocking Fc receptors by incubating for 20 minutes with Fc blocking reagent (Miltenyi Biotec), the cells were separated into aliquots containing at least 5x10⁶ cells per tube and stained separately. Each aliquot was stained with a panel of antibodies or isotype matched controls for 30 minutes on ice. An antibody to CD45 conjugated with APC-Cy7 (1 mg/ml, 1:200), was used to identify leukocyte populations and separate them from remaining renal cells. In the case of CD68 the cells were fixed with 3.5% paraformaldehyde for 10 minutes at room temperature and then permeabilized using BD Perm/Wash™.

FACS acquisition was performed with BD LSR Fortessa equipped with 4 lasers (405nm, 488 nm, 561 nm, 640 nm) and at least 3x10⁶ events were recorded in order to detect rare cell populations. FlowJo software (Tree Star Inc, Ashland, USA) was used for FACS analysis and documentation. The isotype control antibody were matched to the host species and fluorophores used for the staining, and were used alone (i.e. for assign the CD45 gate) and in combination with our panel of staining antibodies used to identify different populations of MPh (for example, CD45 APC-Cy7 + mouse and anti-human isotype APC were used to assign the CD45⁺ CD11c⁺ APC population, S1 Fig)