Propuesta de elaboración de paneles explicativos Primer esbozo prototipo.

2.10.4.1 Cell surface staining (a) Heparanase

Cells were transferred to a 96-well plate (CellStar, U-bottom plate, # 650180; 1 x 105- 106 cells) and washed with FACs wash (200 µl/well) by centrifugation at 389 x g for 3 mins at 4°C. The supernatant was removed, and the cells were incubated with diluted primary human heparanase antibody HP3/17(1.5 µg/ml; see Appendix 1.17). A sample

of lymph node cells (1 x 106 cells/well) was also stained with HP3/17 mAb as a single colour control; FACs wash buffer was added (25 µl/well) to control unstained wells. The plate was incubated on ice for 30 mins. The cells were then washed with FACs wash buffer (200 µl/well) and centrifuged at 389 x g for 3 mins at 4°C. The supernatant was removed, and the cells were incubated with secondary antibody goat anti-mouse Ig PE (2.5 µg/ml; 25 µl/well) for 30 mins on ice in the dark. The cells were centrifuged at 389 x g for 3 mins, washed with FACs wash buffer and vortexed briefly for subsequent staining with leukocyte-specific antibodies (see Appendix 1.17).

(b) Leukocyte markers

To distinguish between sub-populations of leukocytes in the PB and SVFs, the cells were stained with a cocktail of 8 antibodies specific for a range of different leukocyte markers: CD11c, B220, Ly6G, Siglec F, CD45.2, CD11b, CD3, and Ly6C (see Appendix 1.17 for list of fluorochrome-conjugated mAbs). LN cells stained with each individual-conjugated antibody were used for compensation of the spectral overlap between different fluorochromes. The fluorescence was detected using a BD LSR II Fortessa flow cytometer with PE Texas Red, PE Cy5.5, PE Cy7, Alexa Fluor 405, APC, Alexa Fluor 700, APC-Cy7 and Qdot 605 channels. The different leukocyte sub-populations were distinguished based on the cell surface markers are shown in Table 2.1. Events were collected using BD FACS Diva software (version 8). Gating for analysis of heparanase expression on T cells, B cells, dendritic cells, macrophages, neutrophils and eosinophils (see Section 2.10.4.2).

2.10.4.2 Analysis of flow cytometry data

Live cells were distinguished from dead cells based on the forward scatter (FSC) and side scatter (SSC) properties. Single cells were identified using FSC-A and FSC-H and doublets were excluded based on the SSC-W and SSC-H properties. Leukocyte sub- populations were determined based on cell surface markers using the gating strategy in Figures 2.13 and 2.14. The geometric mean fluorescence intensity (GMFI) and geometric mean fluorescence ratio (GMFR; normalised to the Hpse expression of B lymphocytes) of cell surface Hpse (PE) staining on different leukocyte sub-populations was determined using FlowJo software (version 10.0.7, TreeStar Inc., Ashland, OR, USA).

Figure 2.13: Gating strategy for analysing leukocyte sub-populations in peripheral blood of wt mice.

Representative dot plots for peripheral blood show (a) gating of the leukocyte population using forward scatter (FSC) and side scatter (SSC) properties. (b) Single cells were gated based on FSC-A and FSC-H; (c) SSC-W versus SSC-H was used to exclude doublets. (d) The CD45.2+ population was identified and subsequently gated for CD11c+ and CD11c- expression (e). (f) From the CD11c+ cells, conventional dendritic cells (cDCs) were gated using Ly6C-Ly6G- staining; CD11c+Ly6C+ and CD11c+Ly6G+ leukocytes were also identified. (g) The CD11c- population was further divided into myeloid (CD11b+) leukocytes and lymphocytes (CD11b-). (h) The myeloid population was sub-divided into Ly6Cmed-highLy6G+ neutrophils, Ly6ChighLy6G- monocytes and Ly6G-Ly6C- cells. (i) Based on Siglec F fluorescence, the Ly6G-_Ly6C- _{cells were divided into Siglec F}+ eosinophils and Siglec F- _{resident macrophages. (i) Hpse expression on leukocytes (e.g.,} myeloid cells) was shown using histograms of fluorescence staining (blue) versus auto- fluorescence of unstained cells (red). (k) Lymphocytes were identified as B220+ _{B cells} and CD3+ T cells. FSC-A, forward scatter area; FSC-H, forward scatter height; SSC-W, side scatter width and SSC-H, side scatter height.

Figure 2.14: Gating strategy for analysing leukocyte sub-populations in the stromal vascular fraction of wt mice.

Epididymal adipose tissue dot plots show (a) gating of the leukocyte population using forward scatter (FSC) and side scatter (SSC) properties. (b) Single cells were gated based on FSC-A and FSC-H; (c) SSC-W versus SSC-H was used to exclude doublets. (d) The CD45.2+ population was identified and subsequently gated for CD11c+ and CD11c- expression (e). (f) From the CD11c+ cells, conventional dendritic cells (cDCs) were gated using Ly6C-Ly6G- staining; CD11c+Ly6C+ and CD11c+Ly6G+ leukocytes were also identified. (g) The CD11c- population was further divided into myeloid (CD11b+) leukocytes and lymphocytes (CD11b-). (h) The myeloid population was sub-divided into Ly6Cmed-highLy6G+ neutrophils, Ly6ChighLy6G- inflammatory macrophages (INF MP) and Ly6G-Ly6C- cells. (i) Based on Siglec F fluorescence, the Ly6G-Ly6C- cells were divided into Siglec F+ _{eosinophils and Siglec F}- _{resident macrophages. (i) Hpse staining} on leukocytes (e.g., myeloid cells) was demonstrated using histograms of fluorescence staining (blue) versus auto-fluorescence of unstained cells (red). (k) Lymphocytes were identified as B220+ _{B cells and CD3}+ _{T cells. FSC-A, forward scatter area; FSC-H,} forward scatter height; SSC-W, side scatter width and SSC-H, side scatter height.

Table 2.1: Phenotype markers for different leukocyte sub-populations.

Cell type Cell surface marker

Leukocytes CD45.2+

Conventional dendritic cells (cDCs)

CD45.2+ CD11c+ Ly6G- Ly6C-

T cells CD45.2+ CD11c- CD11b- CD3+ B cells CD45.2+ CD11c- CD11b- B220+ Myeloid cells CD45.2+ CD11c- CD11b+

Neutrophils CD45.2+ _CD11c- _CD11b+ _Ly6G+ _Ly6Cmed-high

Eosinophils CD45.2+ CD11c- CD11b+ Ly6G- Ly6Cneg-med Siglec F+ Inflammatory macrophages CD45.2+ CD11c- CD11b+ Ly6G- Ly6Chigh

Resident macrophages CD45.2+ CD11c- CD11b+ Ly6G- Ly6Cneg-med Siglec F- Non-cDC CD11c+ leukocytes: CD11c+Ly6C+Ly6G+