Fluorescence activated cell staining

All protocols in this section were performed using flow cytometric analyses. All cells were acquired on a BD FACSCanto II flow cytometer [BD Biosciences, Oxford, UK] and the post acquisition analysis of flow cytometry data (FCS) files was done using Cyflogic version 1.2.1 software (CyFlo Ltd).

57 2.15.1 Cell surface receptor expression

For DC phenotyping, cells were scraped from petri dishes and washed in HBSS, counted and dispensed into FACS tubes at a density of 1 x 106 cells/tube. Cells were washed with stain buffer (sheath fluid [BD Biosciences, Oxford, UK] supplemented with 2 % FBS and 2 mM EDTA). Cells were then resuspended in 50 µl stain buffer and incubated in the dark at 4 °C for 10 min. Appropriate amounts of CD40FITC, CD86FITC and I-A/I-EPE in combination with CD11cPE-Cy®5.5 were dispensed to each tube in 50 µl stain buffer and tubes incubated for 30 min in the dark at 4 °C. After incubation, cells were washed twice with 4ml sheath fluid and resuspended in 700 µl sheath fluid prior to analyses via flow cytometry.

For splenic, thymic and lymph node T cell phenotyping, cells were processed and dispensed into FACS tubes at a density of 1 x 106 cells/tube. Cell surface staining was performed as above using combination of CD4PE, CD8TC and CD25FITC antibodies. Splenic T cell activation status was also assessed using CD62LFITC, CD69FITC and CD44FITC antibodies in combination with CD4PE and CD8TC.

2.15.2 Dendritic cell endocytosis assay

Endocytic ability of Nrf2+/+ and Nrf2-/- iDCs was determined using DextranFITC as previously described (Sallusto et al., 1995). Harvested iDCs were resuspended at a density of 1 x 106 cells in 100 µl CM and incubated with 0.5 µg/ml DextranFITC (40,000 MW) for 60 min at 37 °C or 4 °C (as negative control). Cells were washed twice in stain buffer, cell surface staining for CD11cPE-Cy®5.5 was performed as above and analysed via flow cytometry.

2.15.3 Dendritic cell phagocytosis assays

Nrf2+/+ and Nrf2-/- iDCs were scraped from petri dishes, washed in HBSS, counted and plated out into 24 well plates at a density of 2 x 106 cells per well. Cells were allowed to adhere for 2 h at 37 °C. Cells were washed twice with warm CM in preparation for addition of apoptotic or necrotic cells.

58 To assess phagocytosis of apoptotic cells, processed thymocytes derived from C57BL/6J mice were incubated with 1 µM dexamethasone at 37 °C under 5 % CO2

for 18 h. Dexamethasone induces apoptosis in murine thymocytes (Cifone et al., 1999). The proportion of apoptotic thymocytes was determined by staining with FITC Annexin V. Over 90% of thymocytes stained positive for Annexin V (as illustrated in Figure 3.4). Apoptotic thymocytes were labelled with CFSE (2 μM) for 20 min at 37 °C in the dark. Cells were washed with media to remove non- incorporated dye and then co-cultured with adherent iDCs at a ratio of 2:1 for 2 h at 37 °C.

To assess the phagocytosis of necrotic cells, Jurkat T cells were fluorescently labelled with 0.5 μM CFSE for 30 min at 37 °C in the dark and necrosis was induced via snap freeze in liquid nitrogen. More than 90 % of cells became necrotic as determined by trypan blue exclusion (data not shown). Necrotic cells were co- cultured with DCs as above.

Cells were then harvested, washed with stain buffer and stained with CD11cPE-Cy®5.5 prior to analysis by flow cytometry.

2.15.4 Annexin V assay

The extent of apoptosis on dexamethasone-treated thymocytes was determined using the FITC Annexin V Apoptosis Detection Kit [BD Biosciences, Oxford, UK] according to the manufacturer’s instructions. One of the early events in apoptosis is the inversion of the cell’s phospholipid bilayer resulting in the exposure of phosphatidylserine molecules. These molecules are readily detected by Annexin V antibody (Fadok et al., 1992). Briefly, dexamethasone-treated C57BL/6J thymocytes (1 x 106 cells per FACS tube) were washed with 1X binding buffer, centrifuged and resuspended in 100 µl of 1X binding buffer. Cells were gently vortexed and incubated with Annexin VFITC antibody for 15 min at RT in the dark. Cells were then washed with 1X binding buffer, centrifuged, resuspended in binding buffer and analysed via flow cytometry.

59 2.15.5 Interferon-γ intracellular staining for DC mediated re-stimulation of F5 CD8 T cells

Immature DCs derived from Nrf2+/+ and Nrf2-/- mice were scraped from petri dishes, washed in HBSS and counted. Immature DCs were pulsed with or without NP68 (1 x 10-7 M) for 2 h at 37 °C. Cell were then extensively washed to remove unbound NP68 and plated out in a 6 well plate (300,000 DCs per well). Cells were allowed to adhere for 2 h at 37 °C. Processed F5 CD8 T cells were co-cultured with the adherent iDCs at a density of 7 x 106 cells per well for 72h at 37 °C under 5 % CO2.

These F5 CD8 T cells were then re-stimulated in 24 well plate with mature Nrf2+/+ DCs (treated with 1 µg/ml LPS overnight) which were either pulsed or unpulsed with NP68 (1 x 10-7 M). This co-culture was at a density of 1 x 106 F5 CD8 T cells and 50,000 Nrf2+/+ mDCs per well. Cells were co-cultured for 6 h in total. Golgi plug inhibitor Brefeldin A (contained within Cytofix/Cytoperm kit as stated below) was added 1 hour after start of culture to allow intracellular cytokine accumulation. Cells were then stained with CD8TC antibody as per 2.15.1 method. Intracellular cytokine staining for IFNγ was achieved using the BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit with BD GolgiPlug™ [BD Biosciences, Oxford, UK] according to manufacturer’s instructions. After cell surface staining, cells were washed with stain buffer and fixed & permeabilised in one step using 1x BD Cytofix/Cytoperm solution for 20 min at 4 °C in the dark. Cells were washed twice with 1x BD Perm/Wash™ buffer and incubated with IFNγFITC plus 1x BD Perm/Wash™ buffer for 30 min at 4 °C in the dark, followed by two final washes with 1x BD Perm/Wash™ buffer and resuspended in 2-4% paraformaldehyde (PFA). Flow cytometric analysis was performed the following day

2.15.6 Effector T cell IFNγ intracellular staining

Day 6 Nrf2+/+ and Nrf2-/- effector T cells were re-stimulated in 24 well plate at a density of a density of 1 x 106 T cells per well with plate bound anti-mouse CD3 (2 µg/ml) and CD28 (2 µg/ml) antibodies for 6 h. Unstimulated cells were used as a background control. Golgi plug inhibitor Brefeldin A (contained within Cytofix/Cytoperm kit as stated above) was added 1 hour after start of culture to

60 allow intracellular cytokine accumulation. Cells were then stained with CD8TC antibody as per 2.15.1 method. IFNγintracellular staining was performed as above. 2.15.7 Effector T cell IL-17A/IFNγ intracellular staining

Day 6 Nrf2+/+ and Nrf2-/- effector CD4+ T cells were re-stimulated in a 24 well plate at a density of 1 x 106 T cells per well either under non-polarizing or Th17 polarizing conditions with 50 ng/ml PMA, 1 µg/ml ionomycin and plate bound anti-mouse CD3 (2 µg/ml). Cells that were left without stimulation were used as background control. Golgi plug inhibitor Brefeldin A (contained within Cytofix/Cytoperm kit as stated above) was added 1 hour after start of culture to allow intracellular cytokine accumulation. Cells were then stained with CD4R-PE antibody as per 2.15.1 method. Interferon-γ intracellular staining was performed as above with addition of IL- 17APerCp-Cy5.5 antibody.

2.15.8 Measurement of reactive oxygen species

Processed Nrf2+/+ and Nrf2-/- splenocytes were washed with HBSS and resuspended in serum-free RPMI 1640. Subsequently, cells (5x 105 cells/ tube) were treated with or without H2O2 for 10 minutes, washed and resuspended in HBSS without phenol

red. The ROS indicator dihydroethidium (DHE) (10 µM) was then added to the appropriate tubes and incubated at 37°C for 10 minutes. Dihydroethidium is a colourless substance that can freely diffuse across the cell membrane. Upon oxidation with intracellular superoxide radials specifically, DHE forms into an oxidised red fluorescent product which is easily detected by for flow cytometric analysis (Burnaugh et al., 2007). Immediately after DHE incubation, cells were analysed on a flow cytometer.