WT or KO-Sirt7 mice were divided into sham surgery or bilateral renal ischemia (22.5 min) groups. The cellular infiltration, particularly macrophages and lymphocytes, were analyzed 72 h post-ischemia because the greatest inflammatory cell infiltration had been reported at this point. [47 (link),48 (link)]. The mice were anesthetized with sodium pentobarbital (30 mg/kg,) and their kidneys were perfused with 20 mL of PBS. One kidney was removed and placed in PBS where it was cut into small pieces with scissors. Collagenase 1 mg/mL and DNAse 50 μg/mL were added and incubated for 30 min at 37° with shaking. A 70 μm cell strainer was used to obtain cell suspension from the digested kidneys. Red blood cells were lysed with ACK lysis buffer. Kidney cells were suspended in PBS to perform a cell count and staining for flow cytometry. The following antibodies were purchased from Tonbo (Tonbo biosciences, San Diego, CA, USA): Ghost dye (violet 510 or APC-Cy7), CD45.2 (APC-Cy7 or APC), F4/80 (VF450), CD11b (PECy7), Ly6G (PE), TCRβ (FITC), CD4 (PerCPCy5.5), CD8 (PE) (Tonbo), and CD206 (APC) (BioLegend, San Diego, CA, USA). All these stains were performed in the presence of Fc block (Tonbo). The samples were read on a NovoCyte Flow Cytometer, Volt Technology (Agilent) and the results obtained were analyzed with NovoExpress software (Agilent, Santa Clara, CA, USA).
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