Anti cd45 alexa fluor 488 clone 30 f11

The following antibodies were used in a surface panel cocktail for the Bl-Eng2 peptide experiments: anti-CD45–Alexa Fluor 488 (clone 30-F11; Biolegend, catalog no. 103122), anti-major histocompatibility complex (MHC) class II tetramer–phycoerythrin (PE), anti-CD4–BUV395 (clone GK1.1; BD, catalog no. 563790), anti-CD8–peridinin chlorophyll protein (PerCP)-Cy5.5 (clone 53-6.7; Biolegend, catalog no. 100734), anti-CD44–BV650 (clone IM7; Biolegend, catalog no. 1033049), anti-CD11b–allophycocyanin (APC) (clone M1/70; Biolegend, catalog no. 101212), anti-CD11c–APC (clone N418; Biolegend, catalog no. 117310), anti-NK1.1–APC (clone PK136; Biolegend, catalog no. 108710), and anti-B220–APC (clone RA3-62B; Biolegend, catalog no. 103212).

Cells from spleen, LNs and BM were counted by trypan blue exclusion under light microscope, after lysis of Red Blood Cells (RBCs) (Sigma-Aldrich). Residual RBCs —identified as Ter119⁺ CD45^— cells by staining with anti-CD45 Alexafluor 488 (clone 30-F11, Biolegend) and anti-Ter119 PerCP-Cy5.5 (clone TER119, eBioscience), and flow cytometry analysis— were found after RBC lysis in spleen (on average 40%) and BM (on average 30%), but not in LNs. Thus, spleen and BM cell counts were multiplied by 0.6 and 0.7, respectively, to obtain nucleated cell counts. Mouse White Blood Cell (WBC) counts/µl and total blood volume were previously reported (21 (link), 22 (link)). The absolute numbers of gag-specific CD8 T cells in spleen, LNs, BM and blood, and of IFN-γ⁺ CD8 T cells in spleen and BM, were estimated based on their percentages determined by flow cytometry and on nucleated cell counts of corresponding organ as previously described (23 (link)).