Validación y confiabilidad de instrumento Instrumento lista de cotejo

2:

Materials and Methods

The general methods and materials which have been employed throughout this study are described in this section. Techniques which were more specific in their application are detailed within the relevant chapters.

2.1:DrosophilaPrimary Embryonic Cultures

The method of homogenising large numbers ofDrosophilaembryos and successfully growing and characterising the differentiated cellsin vitrowas based on a technique called the ‘column drop’ method was originally devised by Shields and Sang (1970). The following method is based on the modified column drop method (Shields et al., 1975).

2.2:Medium

Primary embryonic cultures were grown in a Shields and Sang modified MM3 medium (Shields and Sang, 1977), the constituents of which are listed in the appendix. The medium was further modified for growth of the cells through the addition of 10 % foetal bovine serum (FBS). The medium plus additive was filter sterilized through a 0.22µm filter before use and is referred to as primary (1°) medium.

2.3:Egg collection

Wild-type Oregon-S flies from 7-21 days of age were used as controls for this procedure. The flies, which were kept in food bottles at 25°C, were transferred to a special egg-laying plastic bottle. A square of agar jelly was placed on a watch-glass, and the square smeared with a paste of ground, autoclaved dried

yeast and water. The watch-glass was used to cover the bottle which was then inverted to stand on the watch-glass and incubated at 25°C for 1-2 hours. The watch-glass was removed and these eggs discarded, this is termed the pre-collection. The pre-collection was discarded as it may contain retained embryos further on in development than desired. The watch-glass was

replaced with a fresh one, which was left for 1-2 hours. Eggs were sometimes collected for a short a time as possible so that all the embryos were

comparable in terms of development. Several bottles were used at one collection to ensure adequate numbers of eggs were collected and the eggs were incubated at 25°C until the required stage for processing was reached.

2.3.1:Washing and sterilisation of the eggs

Saturated sugar solution was used to wash the eggs, and as little of the yeast as possible, off the agar squares at which point the eggs float and the yeast sinks. Examination of the embryos floating on the surface of the sucrose solution was made under a dissecting microscope and any hatched larvae were removed. Repeated washing was carried out until all trace of the yeast had been removed. The eggs were finally rinsed with distilled water and transferred into a glass boiling tube with freshly prepared and filtered 3% calcium hypochlorite solution. The eggs were coated with the calcium

hypochlorite solution by inversion and left for 7 minutes, mixing occasionally. Aseptic technique was employed from this point onward. The eggs were poured into a sterile 100µm sieve and washed with plenty of sterile distilled water, then rinsed with 5 mls of primary (1°) embryonic medium. The eggs were then poured into a sterile homogeniser tube and rinsed with 5mls of fresh medium, allowed to settle and most of the medium removed. This was repeated twice more and finally 2ml fresh 1° medium added. Homogenisation was carried out by inserting a pestle slowly into the tube twisting at the

bottom and withdrawing. This was repeated once or twice until no whole embryos or large clumps of tissue were visible. The suspension was pipetted into a sterile universal and centrifuged at 1600rpm for 1 minute, the

supernatant was removed and the pellet resuspended in 2ml fresh 1°

medium. This step was repeated twice more. The pellet was resuspended in 1ml 1° medium, a haemocytometer count was carried out and sufficient medium was added to the remaining 0.9ml to give a final suspension of 3.5 x 106cells/ml.

2.3.2:Column drops

Sterile drilled slides (figure 2.1) were prepared by puttingpetroleum jelly around the well. A 50µl drop of suspension was aliquoted onto a sterile 24x24 coverslip and a drilled slide was inverted with the well placed over the

suspension so a column drop was formed. Slides were left inverted for up to 1 hour to enable cells to adhere to the coverslip. Once turned over the cells become suspended into the medium and grow in a three-dimensional network. The cultures were examined, the slides labelled with date and number and incubated at 25°C in a 5% CO2atmosphere.

Figure 2.1: Column drop slides (Shields and Sang, 1975)

2.4:DrosophilaImaginal Disc Cell Lines (Clone 8+)

The cell lines used in these studies wereDrosophilaClone 8+ (Cl.8+) cell lines which were originally derived from late third instar imaginal wing discs dissected fromDrosphilalarvae (Currie et al., 1988).

2.4.1:Medium

Clone 8+ cell lines were grown in Shields and Sang modified MM3 medium (Shields and Sang, 1977) the constituents of which are listed in the appendix. The medium was further modified for growth of the cells through the

addition of 2% heat inactivated foetal bovine serum (FBS), 12.5IU/100ml insulin and 2.5% fly extract (FE2). The medium plus additives was filtered sterilized through 0.22µm filter and was referred to as complete sterile medium (CSM).

2.4.2:Cell lines

The cell line, Clone 8+ (Cl.8+), and derivatives of the Clone 8+ line which were used in this study are listed in the table below (table 2.1).

Table 2.1:List of Clone 8+ cell line and derivatives with passage numbers. Passage number

30 refers to the age at which the cell line was cloned and the numbers in brackets are the number of passages past cloning.

Name Characteristics Passage number Comments YCl.8+ 20-HE responsive 30 (20-24) Young OCl.8+ 20-HE responsive 30 (70-134) Old

Cl.8R 20-HE unresponsive 30 (21-24) Resistant to ecdysone ZfeCl.8+ 20-HE responsive 30 (68-72) No FE2 in medium

2.4.3:Routine sub-culture of imaginal disc cell lines

Clone 8+ cells were passaged once every 7 days when in culture. Passaging is a term which denotes the splitting of the cells once confluence is reached and before overgrowth, usually on a weekly basis. Cells were pipetted off the tissue culture dish surface and centrifuged at 1200rpm for 5 minutes. The supernatant was removed and the pellet resuspended in 1ml of fresh CSM. Cells were normally seeded at a density of 3 x 106per 5ml CSM onto a 50mm

Petri dish. Dishes were dated and marked with the passage number and cell line name and placed in a humidified incubator with a 5% CO2 atmosphere and set at 25°C. In most laboratories it is not usual practice to take note of the passage number ofDrosophilacell lines. The passage number is often taken as an indication of their‘in vitro’age (Cottam and Milner, 1997). For long term storage Clone 8+ cells were preserved in a liquid nitrogen freezer.

2.5:DrosophilaImaginal Disc Dissection

The imaginal disc structures dissected in these studies and used as controls were taken from Oregon-S late third instarDrosophilalarvae. The dissection technique and culturein vitrowas based on a technique previously described by Milner and Sang, 1974.

2.5.1:Medium

Imaginal discs were cultured in Shields and Sang modified MM3 medium (Shields and Sang, 1977) the constituents of which are listed in the appendix. The medium was further modified for growth of the discs through the addition of 2% heat inactivated foetal bovine serum (FBS). The medium plus foetal bovine serum was filtered through a 0.22µm filter to sterilize.

2.5.2:Micro-Dissection Technique

All dissections took place in a sterilised U.V. hood with a microscope window for a Wild M5 dissecting microscope. All dissecting instruments were

swabbed with alcohol and flamed before use. Cavity slides were pre-prepared by applyingpetroleum jellyaround the cavity. Drops of culture medium were placed onto a siliconised slide and sterile 3rd_{instar larvae placed into a drop to}

wash. Larvae were dissected, the imaginal discs removed and placed into a single drop. Each disc was placed onto a coverslip in a 5µl drop of medium or medium with 0.1µg/ml 20 hydroxyecdysone (herein referred to as ecdysone). A cavity slide was lowered gently onto the coverslip, the medium and disc becoming suspended between the coverslip and the bottom of the cavity slide. The discs were viewed immediately or placed in the incubator at 16°C

overnight until the stage of development required. Further experimental procedures were carried out, such as fixation and antibody staining, which are described with the appropriate chapter.

2.6:Fly lines

Transgenic fly stocks used in many these studies, unless stated, were obtained from the Bloomington stock centre (http://flybase.bio.indianna)

2.7:Moulting hormone (ecdysone)

The hormone used in all the Clone 8+ cell line experiments and imaginal disc dissection was the insect moulting hormone, ecdysone (20-hydroxyecdysone Northern Biochemical Company). For experiments using 10ng/ml of

ecdysone:100μg ecdysone was weighed out onto a foil boat, 5 ml of the appropriate medium added (under sterile conditions) and serial dilutions

made. Filter sterilisation was not carried out as this appeared to affect the activity of the hormone in cultures.

2.8:Microscopy

During routine observations cells were monitored and photographed using a Leitz Diavert photomicroscope or a Nikon inverted photo microscope. Cells treated for visualisation by immunofluorescence were examined using a Zeiss (Oberkochen Ltd) Axioplan 2ie microscope fitted with accessories for indirect fluorescence, differential interference contrast, and phase microscopy. Images were captured using x10, x20, x40, x63 (NA 1.4) oil immersion, Planapo

objectives, and an Orca ER cooled CCD camera (Hammamatsu Photonics UK Ltd). Analysis and image management were conducted using OpenLab

software version 3.09 (Improvision Co. Ltd) and Adobe photoshop version 5.5 (Adobe Systems Incorporated) for Apple Mackintosh. Figures which are found within the Introduction, Materials and Methods and Discussion sections have been given a numerical prefix and suffix. Figures which are listed in the Results sections have been given a numerical prefix and an alphabetical suffix.

Chapter 3