Efluor450 conjugated anti f4 80 clone

To assess granulocyte and macrophage infiltration in sham-operated in IRI-injured kidneys, single cell suspension was created with GentleMacs C-tubes (Miltenyi Biotec) in the presence of 10 mg/mL collagenase IV (Sigma) and 200 U/mL DNase I (Roche) dissolved in Hank’s balanced salt solution. Dead cells were excluded from the analysis using Fixable Viability Dye eFluor 660 (eBioscience). Granulocytes and macrophages were stained with PE-conjugated anti-Ly6G (clone: 1A8, Beckton Dickinson) and eFluor450-conjugated anti-F4/80 (clone: BM8, eBioscience) antibodies, respectively. Samples were analyzed on FACSCanto II flow cytometer (Becton Dickinson). Data analysis was conducted by FlowJo (TreeStar) software.

Granulocyte and macrophage infiltration was assessed in sham–operated and IRI–injured kidneys. GentleMacs C-tubes (Miltenyi Biotec) in the presence of 10 mg/mL collagenase IV (Sigma) and 200 U/mL DNase I (Roche) dissolved in Hank’s balanced salt solution were used to obtain single cell suspensions. Fixable Viability Dye eFluor 660 (eBioscience) was used for detection of dead cells. Granulocytes and macrophages were stained with phycoerythrin (PE)–conjugated anti–Ly6G (clone: 1A8, Beckton Dickinson) and eFluor450–conjugated anti–F4/80 (clone: BM8, eBioscience) antibodies, respectively. Samples were analyzed on a FACSCanto II flow cytometer (Becton Dickinson). Data analysis was conducted by FlowJo (TreeStar) software.