Additional file 5 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 5. Gating strategy for identification of leukocytes and ER adherence to leukocytes with panel 1 in whole blood cultures by identification heterophils, monocytes, and thrombocytes, through singlet gating, FSC/SSC characteristics and using CD45-PerCpCy5.5, CD41/61-Fitc, MRC1L-B-RPE and ER labelled with Far Red (ER-FarRed). From all events in initial dot-plot in A) gating through FSC-H vs FSC-A was performed in B) 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 C). From the CD45 gate in C) events were defined according to FSC and SSC characteristics in D) and high SSC events were defined in P1. P1 events were defined according to CD45 expression in E) and high CD45 expressing events were defined in P2. P2 events were defined according to FSC and SSC characteristics as heterophils in F). Heterophils defined in F) were assessed for ER adhesion in G). Heterophils from the same hen in a control culture incubated without ER-FarRed are shown in L. From the CD45 gate in C) 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). Monocytes defined in I) were assessed for ER adhesion in J). Monocytes from the same hen in a control culture incubated without ER-FarRed are shown in M. Thrombocytes defined in H) were assessed for ER adhesion in K). Thrombocytes from the same hen in a control culture incubated without ER-FarRed are shown in N

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