Additional file 3 of Immune parameters monitored during the production period of laying hens managed with or without single-dose vaccination against erysipelas

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Additional file 3. Gating strategy for enumeration of leukocytes with panel 1 in whole blood samples by identification of counting beads, heterophils, monocytes, thrombocytes, and lymphocytes through singlet gating, FSC/SSC characteristics and using CD45-PerCpCy5.5, CD41/61-Fitc, and MRC1L-B-RPE. From all events in initial dot-plot in A) counting beads were identified as high fluorescent in B). From all events in A) gating through FSC-H vs FSC-A was performed in C) to identify singlets. From this gate high CD45 expressing events (leukocytes) and low SCC-A and medium to high CD45 expressing events (potential thrombocytes) were gated in D). From the CD45 gate in D) events were defined according to FSC and SSC characteristics in E) and high SSC events were defined in P1. P1 events were defined according to CD45 expression in F) and high CD45 expressing events were defined in P2. P2 events were defined according to FSC and SSC characteristics as heterophils in G). From the CD45 gate in D) events were defined according to CD41/61 and MRC1L-B expression in H) and high CD41/61 events were defined as thrombocytes. MCR1L-B positive events from H) were defined according to FSC and SSC characteristics as monocytes in I). Non-thrombocyte events defined in H) were defined according to FSC and SSC characteristics as lymphocytes in J). A representative blood sample from an unvaccinated 50-week-old hen is shown. The antibody panel is described in Additional file 2

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