Quantifying Human Leukocyte Reconstitution

Human leukocyte reconstitution was assessed by flow cytometry using Trucount (BD Biosciences, San Jose, CA, USA) enumeration to calculate the absolute number of human B cells, CD4⁺ and CD8⁺ T cells, monocytes, NK cells, and neutrophils per μL of blood [35 (link)]. Anti-human CD45 (clone H130, BD Biosciences, San Jose, CA, USA) and anti-mouse CD45 (clone 30-F11, BD Biosciences, San Jose, CA, USA) antibodies were used to differentiate mouse from human leukocytes. Human CD45⁺ cells were phenotyped using antibodies specific for human CD3 (clone UCHT1, Beckman Coulter, Brea, CA, USA), CD4 (clone RPA-T4, Biolegend, San Diego, CA, USA), CD14 (clone TüK4, Invitrogen, Carlsbad, CA, USA), and CD19 (clone HIB19, Biolegend, San Diego, CA, USA). Data analysis was performed using FlowJo software (v.9.9.4, FlowJo LLC, Ashland, OR, USA).