Teorías de la situación y de la contingencia del liderazgo

2.2.1 Liposomal transfection of DNA into cells

Twenty four hours before transfection, cells were seeded onto tissue culture dishes, at a density of 1 x 104 cells/cm2. If required for microscopy, the cells were seeded onto 22mm2 _{glass coverslips. The cells were transfected when 50%} confluent as a monolayer, using Lipofectamine Reagent (Invitrogen UK) at a ratio of 1µg DNA to 4µg Lipofectamine. Lipofectamine and DNA were mixed in serum free OptiMEM (Gibco) and incubated together at room temperature, light protected, for 30min. The cells to be transfected were washed and covered in serum free OptiMEM. The DNA Lipofectamine mix was added dropwise to the cells and then incubated for 5-8hrs. The medium was then replaced with DMEM 10% FCS plus supplements as required, and the cells incubated for a further 18- 24hrs. For live imaging, Willco 35mm, glass bottom tissue culture dishes with lids were used.

2.2.2 Making stable expressing mammalian cell lines.

Stable cell lines were made by transfecting 75cm2 vented tissue culture flasks, when 50% confluent, with 10µg of the relevant DNA (with G418 resistance) per flask, using the Lipofectamine protocol described above and in accordance with the manufacturer’s instructions. Cells were grown for 48hrs after transfection in their normal growth medium. Medium was then supplemented with 1mg/ml G418-sulfate and the cells were maintained in this medium thereafter.

2.2.3 Cloning out stable cell lines expressing ABAD-EGFP.

Stable cell lines were cloned out by plating cells at low seeding densities (typically 10 to 50 cells) onto 60mm tissue culture plates. After 10-14 days, plates were imaged and plates with individual colonies were chosen. Selected colonies were enclosed and separated from the rest of the plate by placing a sterile stainless steel cloning ring around the colony. The individual colonies were trypsinised (as described above) and grown in individual wells in a 96 well tissue culture plate to 80-90 % confluency. Transfection was checked by immunofluorescent imaging for 95% cells expressing ABAD-EGFP and western blot for confirmation using anti ABAD antibody.

2.2.4 Culture of multipotent neurospheres and immunocytochemistry

(Carried out in the laboratory of Professor Alessandra D’Azzo, St Jude Children’s Research Hospital, Memphis, USA.)

Cerebella and subependymal zones were isolated from brains of 3- to 6-day-old newborn mice and dissociated into a single-cell suspension by 5 min incubation in 0.25% trypsin-EDTA (GIBCO) and trituration with glass pipettes. Single-cell dissociates were resuspended in growth medium containing DMEM/F12 supplemented with N2 growth supplement (GIBCO), 5% FBS, 20ng ml-1 epidermal growth factor (EGF), and 10 ng ml-1 basic fibroblast growth factor (bFGF). The cells were then plated on ultra low attachment polystyrene 6-well dishes (Corning Costar) at a density of 100,000 cells ml-1. Cultures were supplemented with fresh growth factors every other day. To analyze their cell composition, the neurospheres were transferred onto glass coverslips, coated sequentially with poly- L-ornithine (10 g ml-1) and laminin (5 g ml-1). The cells

quickly attached and began to differentiate. After an additional 48 hr, attached neurospheres were fixed in PBS containing 4% PFA, washed well, and processed for immunocytochemical analysis with polyclonal antibodies against the astrocyte-specific intermediate filament protein, glial fibrillary acid protein (GFAP), and the mAb against the neuronal marker β-III tubulin. Images were analyzed with the confocal laser scanning microscope (Leica, TCS-NTSP).

2.2.4.1 Differentiation of neurospheres

Wild-type neurospheres were trituration with glass pipettes to a single cell suspension and transferred onto Petri dishes coated sequentially with poly-L- ornithine (10g ml-1) and laminin (5g ml-1) and grown in DMEM/F12 supplemented with only N2; FBS or EGF was not added to promote neuronal differentiation. Cells were maintained for 2 days in serum-free DMEM and then fixed in PBS containing 4% PFA and processed for immunocytochemical analysis with polyclonal antibodies. Images were analyzed with the confocal laser scanning microscope (Leica, TCS-NTSP).

2.2.5 Fixing cells and mounting for microscopy

The media was removed from the cells. The cells were then washed 3 times with ice-cold PBS, incubating for 5mins for each wash. The PBS was removed and replaced with ice-cold 4% PFA (paraformaldehyde) to cover the cells. The cells were incubated for 20mins and the PFA removed and discarded in accordance with local rules for toxic waste. Next the cells were rinsed 4 times with ice-cold PBS, incubating for 5mins for each rinse. To mount coverslips, the PBS was

removed and the cells air-dried for 5mins. 10 - 20µl mounting fluid was pipetted onto a microscope slide and the coverslips were placed carefully onto the slide. Several mounting fluids were used including Vectashield (Vector Laboratories), and Mowiol (Calbiochem). Mowiol with added anti-fade was the most successful and provided a hard set. DAPI (4’, 6’- diamidino-2-phenylindole), a DNA- binding fluorochrome, was added for nuclear staining, at a concentration of 0.5 µg/ml. Coverslips mounted with Vectashield were sealed at the edges with nail- varnish.

2.2.6 Preparation of Mowiol mounting medium

Mowiol is a high quality mounting medium with good anti-fade characteristics. It hardens and matches the refractive index of immersion oil and is particularly suited for immunofluorescent microscopy. Anti-fade DABCO is added to further retard photobleaching.

Preparation protocol: 2.4g Mowiol 4-88 (Calbiochem) was added to 6g glycerol and stirred briefly with a pipette. 12ml dH2O was added and stirred at room temperature for 1-2 hrs. Then 12ml 0.2M Tris pH 8.5, was added and heated to 50°C for 1-2hr while stirring. When the Mowiol had dissolved, it was clarified by centrifugation at 55g for 15min. DABCO was added to 2.5% (0.72g). DAPI was added at 0.5ug/ml. Bubbles were removed by centrifugation. 1.5ml aliquots were stored up to 2 weeks at 4°C or frozen at -20°C for long-term storage.

2.2.7 Preparation of 4% PFA (paraformaldehyde)

4g PFA was added to 100ml PBS. In a fume hood, it was mixed and heated to approximately 60°C and then 6-7 drops of 10M NaOH was added until dissolved. It was left to cool and then the pH was adjusted to 7.4 with concentrated HCl. The solution was aliquoted and frozen at -20°C.

2.2.8 Permeabilising and immunostaining fixed cells

After fixing the cells, the PBS was discarded. 0.2%Triton X100 in PBS was added to each dish and incubated for 10min at R/T. Each dish was rinsed 4 times with PBS, incubating for 5 mins for each rinse.

The antibodies were diluted in 3% BSA in PBS. The coverslips were placed into a humidified 90mm tissue culture dish, containing a damp tissue and parafilm. The cells were incubated with the primary antibody, by pipetting 100µl onto the coverslip and incubating for 1hr at R/T. The cells were then rinsed 4 times with 5ml PBS, incubating for 5 mins for each rinse. Then the coverslip was returned to the humidified dish. The cells were incubated with the secondary immunofluorescent-antibody, by pipetting 100µl onto the coverslip and incubating for 1hr at R/T. Each dish was rinsed 4 times with PBS, incubating for 5 mins for each rinse. The coverslips were mounted onto slides as above.

2.2.9 Transfection of cells using the Chariot® protein delivery reagent

Chariot (Actif Motif) is provided as a lyophilized powder and reconstituted in sterile PBS at a concentration of 2mg ml-1. Cells were seeded at 2.5-5.0 x 104

The Aβ peptide (4kDa) was diluted in PBS to achieve 100-500ng per transfection

reaction and 200µl was needed per 60mm tissue culture dish giving a final

concentration of ~ 1.0ng µl-1). The Chariot was further diluted 1:10 in sterile

H2O and 20µl was needed per 60mm tissue culture dish. The diluted Aβ peptide

was added to the diluted Chariot and incubated at room temperature for 30min for the complex to form.

To transfect the cells, the medium was aspirated from cells and the cells were washed once with PBS. The Chariot-Aβ complex was added to the cells and the

appropriate amount of serum-free medium added to the plate and rocked gently to ensure even delivery. The cells were incubated at 370C with 5% CO2 for 60min. To prevent the cells deteriorating due to being in a serum starved environment, complete growth medium was added to the cells without removing the Chariot- Aβ complex. No more than 25% v/v of complete growth medium was added to

the cells, as the presence of serum can interfere with the Chariot transfection efficiency. The cells were incubated for a further 30-60min. Cells were fixed and immunostained.