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foreign tumour masses. Zwi then implanted spheroids to investigate their vasculature following attachment to surrounding organs in comparison with those which remained free floating and non-attached (Zwi et al. 1989). We were interested to use this system to investigate the influence of neovascularisation on proliferative and hypoxic characteristics of spheroids. The first experiment was carried out using SaNeo spheroids but subsequent experiments used SaF spheroids.

2.3.4.1 Pilot spheroid implantation

As a pilot experiment, 10 SaNeO spheroids, between 150 and 350pm diameter, were implanted using a 19g needle into the peritoneum of anaesthetised WHT female mice. Three mice were randomly selected every

week for 6 weeks, sacrificed and the peritoneum examined macroscopically

for spheroids or any tumour cell growth. Free floating and attached spheroids were recovered and placed immediately in neutral buffered formalin. Sections were stained with haematoxylin and eosin.

Ten SaF spheroids between 200 and 300pm in diameter were selected and injected using a 19g needle into the peritoneum of CBA female mice. The SaF is a more rapid growing tumour than the SaNeo so 3 mice were examined daily from day 3 to day 10 after implantation. Only attached spheroids were recovered from the peritoneum with a very low yield.

2.5.4.2 Routine SaF spheroid implantation

Optimisation of the technique is described in Chapter 3. Twenty SaF spheroids between 250 to 350pm in diameter were selected from in vitro

cultures in aseptic conditions by a 19g needle into a 1ml syringe. CBA male mice were anaesthetised with metofane and their abdomen wiped with 70% alcohol. The spheroid containing syringe was flicked a number of times to dislodge any spheroids from the syringe wall and the spheroids injected into the left side of the peritoneum. The syringe was then refilled with 0.9% saline and a further injection given to the mouse into the right side of the peritoneum to rinse any remaining spheroids from the syringe and needle.

The spheroids were recovered 7 days later, from cervically dislocated mice, by a central incision to remove the skin which was peeled outwards disclosing any spheroids growing between the subcutaneous tissue and peritoneal wall. Spheroids attached to the peritoneal wall were excised and the peritoneum exposed. The peritoneal cavity and organs were then examined for any attached or free tumour masses, which were

subsequently excised. The fate of the excised spheroids was dependent on the experimental protocol. Spheroids were either fixed in neutral buffered formalin or snap frozen in liquid nitrogen for immunohistochemical staining or placed in PBS in preparation for disaggregation into a single cell suspension (described later).

2.4 FLOW CYTOMETRY

Flow cytometry is a technique which allows the rapid quantitation of several measurements simultaneously on individual particles such as single cells or nuclei. The particles need to be in suspension such that individual characteristics can be measured, using laser light excitation, based on light scatter and fluorescence, either intrinsic or added immunologically. The flow cytometer is now widely used and has numerous applications in medicine and molecular and cellular biology due to its easy use and rapid analysis of samples.

The accuracy of the flow cytometer often relies predominantly on the state of the sample preparation and staining technique not on the performance of the flow cytometer. The prerequisite for good flow cytometry is obtaining a decent single cell suspension. Techniques which can be used vary from mechanical disaggregation with scissors, enzymic disaggregation or lysing of the cells to produce nuclei. The staining method then has to be adjusted to obtain the maximal differentials between positively and negatively stained cells. This is achieved by manipulation of the titre of the staining reagents and using substances to suppress background fluorescence; in the case of antibodies this would be serum from the species in which the fluorescent reagent was raised. In addition it is important to include appropriate controls such that the specific fluorescence of the measurement being made can be accurately quantified.

The main attributes of flow cytometry are its sharpness of measurement, ability to make several measurements simultaneously, its rapidity and quantitative power. These combine to make the technique very attractive in both the experimental and clinical setting.

2.4.1 The FACScan

The FACScan is an automated cell analyzer attached to a computer. It is capable of measuring five optical parameters simultaneously:- forward scatter, side scatter, and three spectral regions of fluorescence. It has three high performance photomultipliers with band pass filters of 530nm (FITC), 585nm (PE/PI) and >650nm (red fluorescence).

The FACScan has an air-cooled 15 milliwatt Argon-ion laser with a single excitation wavelength of 488nm. The optical layout is shown in Figure 2.1. The fluidics system provides a laminar, single-file procession of cells through the optical sensing region. The sample flow rate is a function of sample capillary tube diameter, sample fluid viscosity and differential pressure. The high flow rate is bOplmin'^ and the low flow rate is 12pknm \ this provides a higher resolution as it has a smaller sample core with more precise cell positioning. Particle velocity in the flowcell is 6msec'\

When individual cells enclosed within a pressurized saline solution pass through the orifice of the flowcell five signal pulses are generated simultaneously from the optical detectors. These pulses are first converted to analog electronic pulses, then to digital data by the A /D converters which can then be stored and processed by the computer system. The software provided with the FACScan allows data acquisition, real-time displays, instrument control, status monitoring and file management facilities. Application software includes single and multiple parameter analysis using single or multiple histograms, dot plots and contour plots with statistics, histogram overlays and DNA cell cycle analysis.

2.4.1.1 Fluorochromes

In the flow cytometry experiments described in this thesis the probes for hypoxia and proliferation have been labelled indirectly with a primary antibody followed by secondary antibodies conjugated to fluorochromes, and then counterstained with a DNA stain. The most commonly used DNA stain is propidium iodide. The phenanthridinium, propidium, intercalates between the bases in double stranded nucleic acids, binding

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