Flow Cytometry Analysis of Immune Cells from Spleen and Aorta

Rats were euthanized under anesthesia and single cell suspensions from spleens were obtained by disaggregating spleen tissue between frosted glass microscopy slides. Erythrocytes were lysed by suspending splenocytes pellets in a hypotonic solution (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA), nucleated cells were washed twice with phosphate buffered saline (PBS). Flow cytometry was performed as described earlier (Singh et al., 2017 (link)). Briefly, washed splenocytes were suspended in Fc blocking buffer (2% v/v fetal bovine serum and 1% v/v normal mouse serum in PBS). An aliquot of 10⁶ cells was taken from each sample and mixed with FITC-CD161, PerCP-CD8a, APC-CD4, PE-CD3, and PE-Cy5-CD45RA antibodies (1 μl each antibody, all antibodies from BD Biosciences). After incubation with antibodies on ice for 30 min, cells were washed twice with PBS and resuspended in 400 μl PBS and flow cytometry was performed on a Beckton-Dickinson Aria flow cytometer. Thoracic aorta were minced with a razor blade and digested in 0.05% w/v Collagenase Type I and 0.05% w/v Collagenase Type II in HBSS. Cells were dissociated by trituration and filtered through a nylon membrane followed by two washes with ice cold PBS. Cells were then processed as described above for the spleen cells.