Ghost dye

CNS tissue homogenates were incubated in digestion medium containing 0.5 mg/mL collagenase type I (Worthington Biochemical) and 1000 U/mL DNase I (Sigma-Aldrich) for 45 minutes following myelin debris removal using a Percoll gradient. LNs were incubated in digestion medium RPMI supplemented with 0.1 mg/mL DNase I (Invitrogen) and 0.1 mg/mL Liberase (Roche). Collected single-cell suspensions were filtered. Resulting cell suspensions were incubated with the following antibodies: anti-CD45 (clone 30-F11, Thermo Fisher Scientific), anti-Ly6G (clone 1A8, BioLegend), and anti-Ly6C (clone HK1.4, BioLegend). Dead cells were excluded using Ghost Dye (eBioscience). For cytokine analysis, LNs were cultured in complete medium (RPMI media containing 10% FCS, supplemented with l-glutamine and antibiotics) with 50 ng/mL PMA and 500 ng/mL ionomycin (Sigma-Aldrich) in the presence of GolgiPlug (BD Biosciences) for 4 hours followed by FACS staining. Cells were stained with Ghost Dye, CD4 (clone GK1.5, BioLegend), IL-17 (clone TC11-18H10.1, BioLegend), IL-10 (JES5-16E3, BioLegend), and IFN-γ (XMG1.2, BioLegend). To assess T cell differentiation, in vitro differentiated CD4⁺ T cells were stained with Ghost Dye, CD4 (clone GK1.5, BioLegend), and IL-17 (clone TC11-18H10.1, BioLegend). Samples were acquired with BD LSRFortessa and analyzed using FlowJo software (Tree Star).

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).