Fig 1.
The Exudate Collecting Chamber (ECC) Model.
(A) The technique employed in the ECC Model: 1&2: Blisters were formed by mechanical suction. 3: Formed blisters. 4: Blisters de-roofed to create skin windows. 5: Silicon ‘Exudate Collecting Chamber’. 6: ECC positioned over skin windows and kept in place by specially designed strap. 7: 50% serum introduced into chamber and then sealed with plugs. (B) Cell counts per ECC chamber achieved with different conditions during model optimisation (50% autologous serum used throughout). Mean ± SD displayed.
Fig 2.
Flow Cytometry Gating Strategy
Representative dot plots of flow cytometry gating strategy for activation markers (FSC, SSC, CD16-APC, CD11b-PerCPCy5.5, CD88-PECy7, CD54-FITC, CD62L-BV421). Exclusion of debris followed by exclusion of doublets. Exclusion of lymphocytes on FSC/SSC. Division of remaining cells by SSC and CD16 into neutrophils (SSCinter-hiCD16hi) and mononuclear cells (SSCloCD16lo-hi). Mononuclear cells were then further subdivided by CD11b into monocytes (SSCloCD11bhi) and dendritic cells (DCs) (SSCloCD11blo). Sample shown using human whole blood.
Fig 3.
Phagocytosis Assay (A) Paired representative examples of flow cytometry gating strategy for phagocytosis assay using AF488-labelled E. coli as described in Methods. Exclusion of debris followed by exclusion of doublets. Selection of neutrophils using FSC/SSC. MFI calculated on single neutrophils. (B) Time-course of ECC neutrophil phagocytosis of AF488 E. coli particles at 5-minute intervals. Gated as in 3A. Expressed as change in MFI from baseline (time 0). (C) 5x106 UVKEc in 50% autologous serum introduced into x2 ECC chambers and x2 chambers used as control. Phagocytosis assay performed after 4 hours of in vivo incubation.
Fig 4.
Cell Count and Composition in the Four Models (A) Total live cell count in each dermal model: ECC (4 hours, 4 chambers pooled), cantharidin (24 hours) and UVKEc-blisters (4 hours). Mean displayed (n = 5/group). Dotted lines represent normal range of number of leucocytes in 1ml of human blood (3-10x106/ml). (B) Percentage of total live single cells making up blister samples split into neutrophils, total monocytes and other cells as per gating strategy in Fig 2. Mean displayed (n = 5/group).
Fig 5.
Neutrophil Cell-Surface Markers
MFI of neutrophil cell-surface markers compared across the different techniques. Mean +SD displayed (n = 5/group). One-way ANOVA with Dunnett’s multiple comparisons test performed across the groups (excluding 4-Hr WB): *p<0.5, **p<0.01, ***p<0.001. ****p<0.0001. Paired t test performed between 4-Hr WB and 4-Hr WB + LPS: ##p<0.01 ###p<0.001.
Fig 6.
All cells gated as shown in Fig 3A to give single neutrophils as defined by FSC & SSC. Results expressed as change in MFI from time 0 to 15-minute time-point. Mean +SD displayed (n = 5/group).