FLOW CYTOMETRY

2.3.1 Double and triple staining of lymphocytes for flow cytometric analysis.

Aliquots of 2x10® cells were stained using the microplate technique (Janossy and Am lot, 1987). Antibody incubations carried out in PBS containing 1% bovine serum albumin (BSA) and 0.02% sodium azide (PBSA) at room temperature for 15-30 minutes, followed by 5 washes in PBSA. Directly conjugated primary antibodies were added at their saturating concentrations, as determined by prior titration. The antibodies used for the staining of

lymphocytes are shown in Table 2.2. In the case of biotin-conjugated antibodies the second layer was a streptavidin-tricolour conjugate (Caltag, San Francisco, USA). Unconjugated primary antibodies were detected using the isotype specific secondary antibodies detailed in Table 2.3. Stained cells were fixed in 1% paraformaldehyde (To prepare 8% paraformaldehyde, 8g

paraformaldehyde was added to 1 0 0 ml PBS and heated to 70°C in a fume

hood for 2 hours. The solution was filtered (0.22pm) and stored at 4°C. This was diluted to 1% strength in PBS before use). Directly conjugated purified mouse immunoglobins of the appropriate isotype were used at identical concentrations to the primary antibodies as negative controls (Sigma).

2.3.2 Flow cytometric analysis of lymphocyte phenotypes.

For each sample, 5000 to 20,000 events were acquired using the Becton Dickinson FACSCAN, and analysed using LYSIS II software. Computer graphics shown in this thesis were generated using WinMDI software. A gate was placed around the lymphocyte population based on forward and side light scatter characteristics prior to analysis. In experiments involving mixtures of fibroblasts and lymphocytes, the T and NK cell population was further identified by expression of the CD2 marker. Most of the data presented in this thesis refers to the percentage of lymphocytes positive for a particular surface antigen. Positive and negative populations for each surface antigen were calculated by the placing of a marker at the point considered to be positive, which was determined by the maximum signal from cells stained with an

irrelevant antibody.

2.3.3 Flow cytometric analysis of fibroblast and endothelial cell surface markers.

Aliquots of 2x10® cells were stained by indirect immunofluorescence using the microplate technique. All staining was carried out in 96 well round bottomed plates (Greiner, Stonehouse, Gloucestershire, UK) which had been siliconized using Sigmacote (Sigma), to prevent cells sticking to the plate. All incubations were carried out in PBSA at 4®C for 1 hour, in a 50pl volume, followed by 5

washes in 200pl PBSA. Cells were first blocked with a Fc fragment of human IgG at lOpg/ml (Calbiochem, Nottingham, UK), in order to prevent possible antibody binding to the CMV induced Fc receptor. Primary antibodies were then added at their saturating concentrations, as determined by prior titration,

followed by the secondary antibodies at their optimum concentration, and fixation in 2% formaldehyde. Purified mouse immunoglobins of specific isotypes were used as controls, at identical concentrations to the primary antibodies (Sigma). The primary and secondary antibodies used are detailed in Tables 2.2 and 2.3. 5,000 to 20,000 events were collected using a logarithmic amplifier and analysed using LYSIS II software. The data was converted from logarithmic values (Mean fluorescence intensity, MFI) to channel values using LYSIS II software, and subsequently to fluorescence intensity units (FlU) for presentation (see below).

2.3.4 Conversion of flow cytometry data to fluorescence intensity units (FlU).

At a resolution of 1024 channels, using 4 log decades, an increase of 256 channels (1024/4) theoretically represents a 10 fold increase in brightness, but in practice this value varies between different machines. Electronic test pulses were used to determine the shift in channel values generated by a 1 0-fold

increase in signal brightness. Briefly, this was achieved by recording the mean fluorescence intensity (MFI) of a given test signal, amplifying the signal by a factor of 1 0 using the manually controlled amplifier on the machine, and

recording the MFI of the amplified signal. This provided a measure of the increase in MFI which represented a 10 fold increase in brightness of the signal, which was then converted to channel values. For our machine, using the FL1 detector at 1024 channels resolution, this value was found to be 235 channels using the above method. The machine could also be calibrated using fluorescent beads of known ‘brightness’, but the disadvantages of fading of fluorescence over a period of time, and discrepancies between different batches of beads led to our decision to use electronic test signals.

For each sample, the data was converted to channel values by the LYSIS II software. Background fluorescence of identical cells stained with an irrelevant antibody were subtracted from the values for the stained samples. The channel values were then converted to fluorescence intensity units (FlU) using the relationship FIU=1 0^ ^ ^ \ where x is the change in median channel value of the

sample from that of the control cells (Watson, 1992). In this study, control cells were uninfected or unstimulated cells stained for the molecule under investigation, and FlU values thus represent fold increases or decreases from

this value. Statistical analysis was performed on these values using the two- tailed Student's t-test.

An example of the method used to calculate FlU for each sample is shown:

Median channel value (class 1 HLA expression)

Median channel value- irrelevant control value

Interferon treated cells 700 600

Uninfected cells 522 422

Change in channels (x) 178

FlU (calculated from 5.72 fold increase

Examination of stained cells by fluorescence microscopy.

In order to check the staining pattern of cells analysed by flow cytometry, aliquots of cells were applied to a poly-L-lysine (Sigma) coated glass microscope slides. Slides were soaked in poly-L-lysine for 1 minute, washed, and allowed to dry in air. Stained cells were applied to the slides and allowed to dry, before mounting in Citifluor® (Citifluor, Canterbury, UK) (PBS-glycerol with p-phenylenediamine to retard fading). Fluorescein labelled cells were examined under an Olympus BH2 fluorescence microscope.

2.4 DETECTION OF CMV ANTIGENS BY