Flow cytometry

8.6.1 Flow cytometric analysis o f M K antigen expression in cell lines

Megakaryocytic phenotypic differentiation was monitored by flow cytometry using a fluorescein isothiocyanate (FITC)-conjugated MoAb Y2/51 that recognises the human glycoprotein GPIIIa (CD61). Cells (1-2 x 10^) were harvested by centrifugation at 200g for 5 min, washed with ice cold PBS containing 1% BSA and then incubated in 0.1 ml PBS/1% BSA with a 1:20 dilution of Y2/51 FITC- conjugated Ab for 1 hr at 4°C. Subsequently, the cells were washed twice with PBS

and finally resuspended in 1 ml PBS. Antigen expression was determined using a FACScan instrument connected to an Apple Macintosh G3 personal computer. Data were recorded and analysed using the Cellquest software programme (Becton Dickinson). Light scatter parameters were used to eliminate dead cells. Relative antigen expression was estimated on live cells using the median fluorescent intensity. An isotype matched FITC-conjugated Ab raised against A. niger glucose oxidase was used as a negative control.

8.6.2 Flow cytometric analysis o f MKs and platelets from liquid cultures o f CD34^ progenitor cells

Platelet and antigenic expression together with MK ploidy were measured by a double-staining technique (Erusalimsky and Martin, 1996). Cells harvested from the migration chamber or taken from cultures directly were washed once with PBS/1% BSA. The cells were incubated in 0.1 ml PBS/1% BSA with a 1:20 dilution of FITC- conjugated Y2/5I anti-human GPIIIA MoAb for Ih. An isotope matched FITC- conjugated Ab raised against A. niger glucose oxidase was used as a negative control. Subsequently the cells were washed with ice cold PBS and centrifuged at 1500 g for 5 min. The cells were then resuspended with I ml of 0.5% paraformaldehyde in PBS and kept on ice for I hr. The paraformaldehyde was diluted with 5 volumes of cold PBS and the cells were centrifuged at 1500g for 5 min. The pellets were finally resuspended in 1 ml of DNA staining solution (see p 100) and left overnight in the dark. All the procedures described above were performed at 4°C. Prior to fixation each centrifugation step was carried out after

Cells were analysed using a FACScan flow cytometer. Data were recorded and analysed using the Cellquest software programme. The set-up of this machine is summarised in Fig. 8.3. Briefly, the instrument was set for the measurement of forward and side scatter and the analyser threshold adjusted on the forward scatter channel (FSC) to exclude subcellular debris. Green (FITC) and red (PI) fluorescence of each cell was detected using the FLl and FL3 channels respectively. Overlaps between emission spectra were corrected electronically. Cells stained with PI alone were used to correct for spillage of the red fluorescence signal into the green fluorescence detector. The sample stained with the two fluorochromes was used to correct the spillage of green fluorescence into the red fluorescence detector. To identify the platelets in the culture instrument settings were adjusted using human washed platelets as a reference. In figure 8.3A, region R1 was defined by running CD61^-FITC labelled human platelets in the flow cytometer. Particles from the cultures falling within this region were then analysed for CD61^-FITC expression relative to the negative control (Fig. 8.3B). A separate region, R2, containing cells was identified based on forward scatter characteristics (Fig. 8.3A) and FL3 (PI)

fluorescence (Fig. 8.3D). Cells falling within this region were again identified according to their expression of CD61^-FITC expression relative to cells labelled with the FITC-control Ah (Fig. 8.3C). Cells falling within R2 that expressed CD61^ were then analysed according to their ploidy distribution as measured by red (PI) fluorescence on the FL3 channel (Fig. 8.3D). Each sample was analysed for 30 seconds at ‘high flow’ during which time all events were collected. The high flow setting on the flow cytometer was calibrated to process 1 pL of cell suspension per second.

B Ml 1 0 ° 1 0 ' 1Q2 103 CD61 FITC 10' Ml 1 0“ 1 0' 103 10“ 10' CD61 FITC A ° D 1 0° 1 0' 1 0“ 1 0“ Forward S catter

Figure 8.3 Analysis of CD34 derived cultures according to CD61 expression and ploidy class.

A R1 identified based on human platelet gate (see text). R2 identified by cell size and FL3 (PI) fluorescence. B & C R1 and R2 analysed according to CD61 expression. The grey overlay shows the fluorescence obtained from cells stained with the negative control. D Cells from R2 that express CD61 analysed according to PI fluorescence on the FL3 channel. Electronic markers (M l, 2 & 3) are demonstrated that allow the quantification o f events according to fluorescence but are specific to each plot.

8.6.3 Quantification o f cell migration

In experiments involving CD34^ cultures and purified MKs, cells were double labelled as described above prior to quantification. In experiments involving M07e and UT-7/mpl cells, quantification was performed without prior labelling. In experiments involving CD34^ cultures or purified MK, analysis gates were established to assess the migration of different cell subpopulations. In each case the proportion of cells migrating was calculated by dividing the number of cells that were recorded from the lower compartment by the sum of the cells recorded from upper and lower compartments and expressed as a percentage. Regular checks were made to ensure that the volume of cells processed by the FACScan instrument during the 30 seconds acquisition period remained constant for all experiments. In some experiments results obtained by flow cytometry were verified by haemocytometric cell counting.

8.6.4 M easurement o f particle size

An important part of this work involves the measurement of platelet size. For the in vivo work, commercially available cell counters were used to measure MPV using an impedance technique. This technique involves the calibration of the measuring apparatus to count the number of cells passing through an electronic aperture based on their size. However this technique was not capable of measuring particle size from the cultures for two reasons. Firstly the concentration of cells in the cultures was too low for accurate counts using clinical settings and secondly the cultures produce a lot of debris of a size similar to platelets that would have been counted by the equipment. The ideal counter would have combined a flow cytometer and coulter cell to allow the measurement of size of fluorescently labelled particles. However this equipment was not available. Therefore, forward scatter as measured by the flow cytometer was used as a measure of particle size. This had the advantage of allowing the measurement of discrete populations of cells based on fluorescent Ab labelling. However, the measurements produced by the flow cytometer were in arbitrary units and relied on the light scatter characteristics of the cells under investigation. These cells (in particular platelet-like particles) have the ability to change shape and surface characteristics (eg when activated) both factors that influence the light scatter characteristics of the cell. Thus measurements of forward scatter allow some idea of

relative differences in size of cells but require careful interpretation based on the morphology of the cell surface.

8.7 Statistical analysis

An unpaired Students T-test was used to assess the statistical differences between the means of data sets using the Graphpad Prism software (version 3.0). Statistical significance was reached with p<0.05.