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Tritiated ^H-thymidine and Na2^’Cr0 4 were both purchased from Amersham

2.2 Methods

2.2.1 Cell Viability Test by Trypan Blue Exclusion Method

A small volume o f cell suspension to be counted (e.g., lOp.1) was mixed with the same

volume o f trypan blue solution (Sigma Chemical). This was then layered on a cell

counting chamber (Weber Scientific International) and viewed under a light

microscope (Leica) at a magnification o f xlOO. Viable cells which had excluded

trypan blue were counted and the number o f the cells in the suspension was calculated

as follows:

Number o f viable cells in 25 squares x 2 (dilution factor) x 10^ = number o f cells/ml

2.2.2 Cryopreservatiou aud Retrieval of Cells

Cells to be cryopreserved were counted, and between 5 x 1 0 ^ and 2 x 1 0 ^ cells were

then resuspended in 1ml FCS with 10% v/v DMSO per vial o f cryotube (Greiner).

These were placed in a freezing container (Cryo 1°C, NALGENE) which allows

gradual decrease in temperature (1°C per minute) and stored at -80°C overnight

before being transferred to the liquid N] storage. For retrieval o f frozen cells,

cryotubes were thawed in 37°C water bath. Cells were then washed twice in cold

supplemented medium before counting and cultured at 37C°.

2.2.3 Bone Marrow DC (BmDC) Preparation from Mice

Femurs and tibias were removed under sterile conditions. Bone marrow was isolated

by removing the ends o f the bone with a pair o f scissors and forceps and flushing out

marrow cells were cultured in BM-CM and lOng/ml recombinant mouse GM-CSF

(R&D Systems) at approximately 10^ cells/ml in 75cm^ tissue culture flasks (TPP).

At day 3 (72 hours), non-adherent cells were removed by replacing the medium

completely with fresh BM-CM with GM-CSF. At day 5 (120 hours), semi-adherent

cells were removed from the culture flask, resuspended in fresh BM-CM at 10^

cells/ml and cultured in 24 well tissue culture plates (NUNC™, Nalge Nunc

International, Denmark) for 24 hours (unless otherwise stated), w ith or without

addition o f modulins (table 2.3).

2.2.4 Immunofluorescent Labelling o f Cell Surface Markers

Cells to be examined were preincubated at 10^ cells/ml in blocking buffer (described

below) for 15 minutes at 4°C before labelling with antibodies to minimise non­

specific binding. PBS containing 0.1% w/v bovine-serum albumin (BSA) (Sigma)

and 0.01% NaN] (Sigma) with 5% v/v normal mouse serum (Sigma) was used as a

blocking buffer for labelling with rat- or hamster-derived antibodies, while 5% v/v

FCS replaced the mouse serum for labelling with mouse-derived antibodies.

Antibodies directed to cell surface markers (section 2.1.8) were diluted in the

blocking buffer. 10^ cells were labelled with 25pi o f the diluted antibodies for 15

minutes at 4°C, followed by two washes with cold PBS containing 0.01% NaN].

Labelled cells were examined by flow cytometry using a FACScalibur (Becton

Dickinson, Oxford, Oxon).

2.2.5 Immunofluorescent Labelling o f Intracellular Cytokines

Following appropriate treatment, cells were further stimulated at 10^ cells/ml in

at 37°C 5% CO2 in order to amplify the cytokine production. After 2 hours o f the 6

hour incubation Brefeldin A (Sigma) was added at a final concentration o f lOpg/ml.

These cells were harvested and incubated with the blocking buffer (section 2.2.4) at

10^ cells/ml for 15 minutes at 4°C. 10^ cells were first labelled with the antibodies

directed to cell surface markers and washed as described earlier. lOOpi PermeaFix^^

(Ortho Diagnostic Systems) was added to the cells (which permealises and fixes the

cells) and incubated at room temperature for 45 minutes in the dark. This was

followed by two washes with cold PBS containing 0.01% NaNg. Cells were then

labelled with 25pl o f diluted antibodies for intracellular cytokines for 30 minutes at

4°C, followed by two washes with cold PBS containing 0.01% NaN]. Cells were

examined by flow cytometry using a FACScalibur.

2.2.6 Labelling Cells with CFSE

Cells to be labelled were resuspended at 5 x 10^ cells/ml in serum-ffee PBS. A lOmM

stock solution o f carboxyfluorescein diacetate succinimidyl ester (CFSE, Cambridge

bioscience, Cambridge, UK) in DMSO was added to a final concentration o f 5pM and

incubated at 37°C for 10 minutes. At the end o f the incubation period, the cells were

immediately washed 3 times in cold BM-CM.

2.2.7 Confocal Microscopy and Image Acquisition

Cells to be examined by confocal microscopy were prepared as described in section

2.2.4. Labelled cells (2x10^) were mounted on microscope slides using cytospin

(Shandon) and allowed to dry. Samples were then examined by laser confocal

microscopy (Leica TCS NT, Leica). Areas o f interest were selected by conventional

objectives. Samples were scanned in a raster fashion to produce an electronic image

o f 1024x1024 pixels. The filter settings were altered manually to match the excitation

and emission spectra o f the fluorochromes. Images in X-Y sections were obtained

from sequential scans in a Z plane.

2.2.8 Peptide Binding Assay

Efficiency o f a peptide to bind MHC class I molecule was assessed using RMA-S

cells (Ljunggren et al., 1990; Schumacher et al., 1990). RMA-S is a murine

lymphoma cell line and expresses H-2D^/K^ stably at 26C° but not at higher

temperatures in the absence o f added exogenous peptide (Schumacher et al., 1990).

However, the expression can be stabilised at 37C° upon binding to a peptide

(Ljunggren et al., 1990; Schumacher et al., 1990). RMA-S cells were cultured at 10^

cells/ml in RPMI-CM. lOOpl o f the cell suspension was plated in each well o f 96

well U-bottom plates, and incubated for 5 hours to overnight at 26°C. 1-2 hours

before the end o f the incubation period, titrated concentrations o f peptides were added

to the cells. The plates were then transferred to 37°C and incubated for 2-8 hours.

Efficiency o f the peptide binding was determined by measuring the expression o f H-

2K molecules on the cell surface by flow cytometry.

2.2.9 Purification o f CD4^ and CDS^ T cells

CD4^ T cells and CDS^ T cells from BALB/c or C57BL/6 mice were isolated directly

by using anti-mouse CD4/8 magnetic columns (Miltenyl Biotec). Briefly, spleen and

peripheral lymph nodes were removed from mice and made into single cell

suspensions in RPMI-CM by filtering through 70pm cell strainers. RBC from the cell

for 10 minutes. RBC were then removed by brief centrifugation to sediment the lysed

RBC. The RBC-depleted cell suspension was washed in RPMI-CM, counted, and

preincubated in PBS containing 0.5% w/v BSA and 5% v/v normal mouse serum for

15 minutes at 10^ cells/ml to minimise non-specific binding o f the antibodies. Cells

were then labelled with anti-mouse CD4/8 magnetic beads and isolated according to

the m anufacture’s recommendation. For cytokine studies in the DO 11.10 transgenic

system, single cell suspensions o f splenocytes made from DO 11.10 transgenic mice

were first positively selected for cells expressing CD62L using the MACS columns as

described above. CD62L positive cells were then stained with PE-conjugated CD4

antibody (H129.19, Pharmingen) and were sorted using the MoFlo sorter (Fort

Collins, Colorado, USA). Purity o f the sorted cells was examined by flow cytometry.