2.2.1
Extraction of mouse bone marrow cells
The femurs and tibias of mice were removed from euthanized animals. The bones were cleaned and sterilised in 70% ethanol. Using MACS buffer containing PBS (Gibco), 2% heat inactivated fetal bovine serum (FBS) (Seralab), 2mM EDTA, the bones were flushed through with 5 ml syringe and 23 G needle (BD Microlance) to liberate the marrow cells. The harvested bone marrow cells were filtered through a 70 µm cell strainer (Fisher) to remove any debris and further washed with MACS buffer, centrifuged at 1600 rpm for 5 min. The supernatant was discarded and cell pellet resuspended in an appropriate volume for counting. To count viable cells, cell suspension was diluted 1:1 ratio with trypan blue (Sigma) for dead cell exclusion and counted using a haemocytometer, visualised by light microscope. The value obtained from one primary 1 mm2 square was equal to
x 107 /ml in sterile PBS for i.v. injections for the generation of chimeras or used
for culturing bone marrow dendritic cells.
2.2.2
Culture of bone marrow dendritic cells (BMDC)
The cells harvested from bone marrow were counted as previously described and resuspended to 2.5 x 106 /ml in complete R10 media and 0.2 µl/ml Flt3 ligand
recombinant protein (Invitrogen). Complete R10 media contained RPMI 1640 (Gibco), 10% FBS, 1% Pen/Strep (Gibco), 1% Glutamine (Gibco), 1% HEPES (Gibco), 1% Sodium Pyruvate (Gibco), 0.1% 2-Mercaptoethanol (Gibco). Cells were seeded at 1 ml/well in a 24 well plate (Costar) and incubated for 9 - 10 days at 37°C, 5% CO2. On day 5 of culture the media was changed, removing existing
media and replacing with fresh complete R10 media and Flt3 ligand.
2.2.3
Immune cell isolation from visceral adipose tissue
Visceral adipose tissue (VAT) was collected from euthanised mice, from the gonadal, epididymal, mesenteric and peri-renal fat depot sites, with care to first remove the lymph nodes. The tissue was weighed and placed into tissue wash buffer containing PBS and 2% FBS and the tissue was diced. The weight was recorded for cell number calculations and to determine the percentage of body weight. To digest the tissue, 5688 U Collagenase II (Sigma) and 0.32 U DNase (Sigma) was added per gram of VAT in 2 ml wash buffer and incubated at 37°C with agitation 220 rpm for 30 min. The digested tissue was passed through 100 µm cell strainer (Fisher) and centrifuged at 2000 rpm for 10 min. If a fatty layer was observed after spinning, the AT was under digested and the layer was collected and re-digested. The supernatant was discarded and the combined pellets of immune cells present in the vascular fraction were isolated. The pellet was lysed for red blood cells using ACK lysis buffer, containing 150mM NH4Cl
(Sigma), 10mM KHCO3 (Sigma), 0.1mM Na2EDTA (Sigma) dissolved in H2O and
pH adjusted to 7.2 - 7.4. The cells were resuspended in 1 ml ACK lysis buffer for 2 min, and washed with tissue wash buffer and centrifuged at 1800 rpm for 5 min. The resulting cell suspension was filtered through a 70 µm cell strainer and centrifuged at 2000 rpm for 10 min. The supernatant was discarded and the cell pellet was washed with PBS. The immune cell pellet was stained for analysis by flow cytometry or placed in Trizol (Invitrogen) for mRNA analysis.
2.2.4
Immune cell isolation from spleen and lymph nodes
Spleen and mesenteric, inguinal, cervical, axillary, brachial or mediastinal lymph nodes were collected from euthanised mice. The spleen or lymph nodes were placed in serum free DMEM (Gibco) and digested with 400 U/ml Collagenase D (Roche) using 5 ml syringe and 25 G needle (BD Microlance) to balloon the tissue to expel cells, followed by fine dicing of the tissue. The spleen or lymph nodes were incubated with the Collagenase D for 18 min at 37°C and digestion was stopped by adding EDTA. The digested spleen or lymph nodes were passed through 70 µm cell strainer and centrifuged at 1600 rpm for 5 min. The supernatant was discarded and red blood cells in the pellet were lysed using ACK lysis buffer and washed with PBS. The immune cell pellet was stained for analysis by flow cytometry or for further cell isolation.
2.2.5
CD11c
+cell and CD4
+T cell isolation
CD11c+ cells and CD4+ T cells were isolated from spleen or VAT by positive
selection using microbeads and LS columns (MACS Miltenyi Biotec). The isolated immune cells were counted and resuspended at 1 x 108 /400µl in MACS buffer.
The cells were incubated with 40 µl CD11c+ or CD4+ microbeads (MACS Miltenyi
Biotec) for 20 min at 4°C. The cells were washed with MACS buffer, centrifuged at 1800 rpm for 5 min and the supernatant was discarded. The cells were then resuspended in 800 µl of MACS buffer and loaded into a pre-washed LS column while being placed in the magnetic field of a MACS separator. The column was washed three times with MACS buffer and the flow through collected was discarded. After the final wash the LS column was removed from the magnet and 5ml MACS buffer was loaded into the column and the cells bound with microbeads within the column were flushed out using the plunger into a new tube. The cells were centrifuged at 1800 rpm for 5 min and the supernatant was discarded. The cells were counted and resuspended in complete R10 media to an appropriate cell concentration for plating.
2.2.6
CD3
+T cell isolation
CD3+ T cells were isolated from spleen and lymph nodes of BALB/c mice by
negative selection using Dynabead (Invitrogen). The isolated immune cells were counted and resuspended at 1 x 108 /ml in MACS buffer. For an initial blocking
step FBS was added to the cells in a 15 ml tube, followed by the antibody mix. After 20 min incubation at 4°C, the cells were washed twice with MACS buffer, centrifuged at 350 g for 5 min and the supernatant was discarded. The cells were resuspended in MACS buffer and incubated with pre-washed Dynabeads for 15 min at room temperature with agitation. After mixing, the tube was placed in the DynaMag magnet for 2 min to allow for separation and while the tube remained in the magnet the unbound CD3+ T cells were transferred into a new tube. The
new tube was placed back into the magnet and the separation was repeated. The CD3+ T cells were washed in MACS buffer, centrifuged at 1800 rpm for 5 min and
the supernatant was discarded. The CD3+ T cells were counted ready for CFSE
labelling.