Cd45ra pe cy5

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.

Approximately 500 μL of venous blood was obtained and stained with mouse anti-human (APC CD3, BD Pharmingen^TM Cat.555335; FITC CD4, BD Pharmingen^TM Cat.555346; and PE-Cy™⁵ CD45RA, BD Pharmingen^TM Cat.555490) antibodies. Briefly, venous blood was diluted with an equal volume of plain RPMI-1640 media and aliquots of 100μL were pipetted into snap-cap tubes. The blood-media mixture was then stained with 10 μL of fluorochromed surface antibodies and incubated at 4°C for 30 minutes. The red blood cells (RBCs) were then lysed using cold 1X BD Pharm Lyse^TM lysing buffer (Cat.555899) at room temperature for 10 minutes. The cells were washed twice in cold wash buffer (5g Bovine serum albumin (BSA) + 0.5g Sodium azide in 500mL Phosphate buffered saline (PBS)) and fixed in 1% paraformaldehyde at 4°C. Cells were then acquired using a four-color FACSCalibur within 24 hours of staining. Lymphocytes were acquired and stored on gate R1 (Fig 1A). These were further gated as CD4+ and CD4- lymphocytes as shown in Fig 1B. In order to determine the proportion of CD4+ lymphocytes that were T cells, a third gate for CD3+ was used (Fig 1C). This gate demonstrated that ~98.0% (median value) of the CD4+ lymphocytes were (CD3+) T cells.

Mononuclear cells were prepared from peripheral blood on Ficoll‐Paque (GE Healthcare) gradients as previously done (Shin et al., 2015). Peripheral blood mononuclear cells (PBMCs) were labeled with antibodies to APC‐cyanin 7 (Cy7)‐CD3, Pacific Blue–CD8α, PE–Cy5–CD45RA, PE‐Cy7‐CCR7 (all from BD Biosciences), FITC‐IL‐7Rα (R&D Systems), and PE‐CX3CR1 (BioLegend). Some cells were fixed and permeabilized using Cytofix/Cytoperm solution (BD Biosciences) and additionally stained with antibodies to PE‐FGFBP2, PE‐GZMB, and biotinylated GZMH (all from R&D Systems). Biotinylated GZMH antibodies were then labeled with Pacific Blue conjugated anti‐goat IgG (Biolegend) secondary antibodies. Stained cells were analyzed using an LSRII® flow cytometer (BD Biosciences) and FlowJo software (TreeStar). For FACS sorting of IL‐7Rα^low and ^high EM CD8⁺ T cells, freshly isolated PBMCs were stained with antibodies to APC‐Cy7‐CD16, Pacific Blue–CD8α, PE–Cy5–CD45RA, PE‐Cy7‐CCR7, and FITC‐IL‐7Rα antibodies. Stained cells were sorted into IL‐7Rα^low and ^high EM (CCR7⁻) CD8⁺ T cells using a FACSAria® (BD Biosciences). The purity of cells was >97%.