Prueba 1: Variación de velocidad de transmisión

2.7.1 The detection of the expression of CMV-specific IE antigen by flow cytometry.

Smooth muscle cells and fibroblasts were seeded at a density o f 1.0 x 10^ cells/well in 12-well plates and incubated for 24 hours. The cells were infected with CMV strain AD 169 or strain C IF at a m ultiplicity o f infection o f from 0.3-10 pfu/cell as indicated in the text. At 24 hours post­ infection, infected cells were removed by trypsinization using trypsin-EDTA (500|ig/ml). The cells were washed twice with phosphate buffered saline by centrifugation at 500g for 5 minutes, and fixed with phosphate buffered saline: acetone (66:34) for 20 minutes at 4°C. The cells were then washed and incubated with a monoclonal antibody specific for CMV IE antigen, designated E l 3, for one hour at 37°C in a humidified chamber. Irrelevant isotype-matched antibodies at the same concentration as the prim ary antibody were used in parallel. The cells were then washed and incubated with a FITC-conjugated F(ab' ) 2 fraction o f a goat anti-mouse IgG antibody at 37°C for 30 minutes. Following washing, the cells were fixed in 2% paraformaldehyde, and analysed

by flow cytometry using a FACScan (Benton Dickinson, Mountain view, CA) as described in section 2.7.4.

2.7.2 The detection of a fibroblast-specific cell surface marker by flow cytometry.

Smooth muscle cells and fibroblasts were seeded into 6-well plates at a density o f 2.5 x 10^ cells per well, and cultured for 24 hours. The cells were trypsinized, washed twice in phosphate buffered saline, and incubated with a fibroblast-specific monoclonal antibody, designated FibAS02, or with an irrelevant isotype-matched control antibody for 1 hour at 37°C. The cells were again washed in phosphate buffered saline, and stained with a FITC-conjugated F(ab’ ) 2 fraction o f a goat-anti-mouse IgG antibody for 1 hour at 37°C. Following incubation, the cells were washed twice with phosphate buffered saline, fixed in 2% paraformaldehyde, and analysed by flow cytometry using a FACScan as described in section 2.7.4.

2.7.3 The detection of the cell surface expression of adhesion molecules and class I and class II MHC by flow cytometry.

Smooth muscle and fibroblasts were seeded into 6-well plates at 3 x 10^ cells/well, and 24 hours after the initial seeding were either left uninfected, or infected with CMV strain AD 169 or strain C IF at a multiplicity o f infection o f 10 pfu/cell. In some experiments, the CM V-infected smooth muscle cells were either stimulated with 10 ug/ml IFN-gamma (Sigma), or left unstimulated. The cells were detached by trypsinization, and washed in phosphate buffered saline containing 0.1% sodium azide (BDH) and 0.1% bovine serum albumin (Sigma). The cells were then stained with monoclonal antibodies specific for certain adhesion molecules, class I or class IIM H C -

determinants, or their appropriate isotype-matched control antibody, at their saturating concentrations (see section 2.5) for 30 minutes. This was followed by staining with FITC- conjugated F(ab’ ) 2 fraction o f a goat anti-mouse IgG antibody for a further 30 minutes. After washing, the cells were fixed in 2% paraformaldehyde (BDH), and subsequently analysed by flow cytometry using a FACscan as described in section 2.7.4.

2.7.4 The acquisition and analysis of flow cytometric data

For all flow cytometric analyses using the FACScan IV flow cytometer, 5000-10000 events were collected using a logarithmic amplifier, and the data obtained was processed using the consort 30 and lysis II software programmes (Becton Dickinson) or the Win M DI version 1.3.3 programme (Verity Software House, Maine, USA). After exclusion o f the dead cells from the original cell

population using the forward and side scatter dot plot profiles, the data from the viable cells was obtained using a logarithmic scale, and presented either as histogram profiles, the percentage of cells positive for the appropriate antigen, or the median fluorescent intensity in arbitrary units. W hen stated in the text, flow cytometric data were converted from logarithmic values (median fluorescence intensity), to median linear channel values, using the lysis II software programme, and subsequently to fluorescence intensity units (FIU). The background fluorescence o f parallel samples stained with an irrelevant isotype-matched antibody was subtracted from the values obtained for the test samples. The median linear channel values were then converted to FIU using the relationship FIU=10^^“^^\ where x is the difference in median linear channel value o f the sample from that o f the control cells (Watson, 1992). The value 235, represents the shift in channel values that generated a 10-fold increase in signal brightness as determined using electronic test pulses on the FACScan. Briefly, this was achieved by recording the median fluorescence intensity o f a given test signal, amplifying the signal by a factor o f 10 using the m anually controlled amplifier on the machine, and recording the median fluorescence intensity o f the amplified signal. This provided a measure o f the increase in median fluorescence intensity, which represented a 10-fold increase in brightness o f the signal, which was then converted to channel values. For the machine used in this study, this value as determined using the FLl detector at 1024 channels resolution was found to be 235 channels. In this study, control cells were uninfected cells stained for the molecule under investigation, and FIU values thus represented fold increases or decreases from this control cell value. Statistical analysis was performed on these values using the M ann-W hitney t-test.

2.8 ORGAN CULTURE OF SAPHENOUS VEINS