Cd8a percp cy5 5 clone 53 6

Flow cytometric analysis was used to assess the purity of adoptively
transferred NK cells prior to transplantation. Cells obtained after NK isolation
(see above) were incubated for 20 minutes at 4°C with
CD3ε-PerCP/Cy5.5 (clone 145.2C11, BioLegend), CD122-FITC (clone TM-
β1, BD Pharmingen), and NK1.1-APC (clone PK136, eBioscience). To detect
the possible presence of CD4+ and CD8+ T cells, a separate cell preparation was
also stained with CD45.2-APC (clone 104, eBioscience), CD3ε-PerCP/Cy5.5,
CD4-Pacific Blue (clone RM4-5, BD Biosciences) and CD8a-PerCPCy5.5 (clone
53-6.7, BD Biosciences). The cells were then washed once with PBS, 1% BSA, and
0.1% sodium azide (FACS buffer); data was collected using a BD LSR II flow
cytometer (BD Biosciences) and analyzed with FlowJo software (Ashland, OR, USA).
This same analysis was also used to detect the presence of these adoptively
transferred cells in recipients post-transplantation. Here, the spleen was
recovered from recipient animals on day 30 and depleted of erythrocytes via use
of red blood cell lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) and
re-suspended in FACS buffer. Cells were incubated with the same antibodies and
analyzed as described above.