Spleen cells of a CD57BL/6 mouse were pulsed with or without the ovalbumin peptide 257–264 SIINFEKL for two hours. Then cells were harvested, washed, and stained with Anti-H-2K^b/SIINFEKL antibodies conjugated to PE (A), APC (B), PE-Vio770 (C), or Biotin (D) as well as with CD11c antibodies and CD45 antibodies and analyzed by flow cytometry using the MACSQuant® Analyzer. Cells labeled with Anti-H-2K^b/SIINFEKL-Biotin were stained with Anti-Biotin-APC. Gates were set on CD45⁺/CD11c⁺ cells. Histograms show pulsed positive cells (right peak), and as negative control unpulsed stained cells (left peak). Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence or 4',6-diamidino-2-phenylindole (DAPI) fluorescence, as in the case of Anti-H-2K^b/SINFEKL-PE-Vio770.