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2.5.1 Cell culture maintenance

Cells were maintained as monolayer cultures unless stated otherwise, at 37°C in an atmosphere of 5% carbon dioxide. Cells were passaged every two to three days or when approximately 90% confluent, except where stated.

2.5.1.1 Suspension cells

Human lymphoblastoid cells corresponding to a large Moorfields pedigree with the Arg120Stop nonsense mutation in RP2 and randomly selected control males were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK). Cells were maintained in suspension culture in RPMI 1640 Glutamax-I (Life Technologies) supplemented with 10% foetal bovine serum (FBS)(Sigma) with media changes every two to three days. No antibiotics were used in the routine culture of these cells.

2.5.1.2 Adherent cells

Human neuroblastoma SH-SY5Y cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK293T) cells and human retinal pigment epithelial ARPE19 were cultured in Dulbecco’s modified Eagle’s Medium (DMEM)/F12 (Life Technologies). Cervical adenocarcinoma cells (HeLa cells), were cultured in DMEM and human intestinal epithelial Caco-2 cells were cultured in DMEM, containing 0.11 mg/ml sodium pyruvate. All cells were cultured in the presence of 10% foetal bovine serum and 50 pg/ml gentamicin, with the exception of ARPE19 cells which were cultured in the presence of 10% FBS and 100 units/ml penicillin and 100 pg/ml streptomycin.

2.5.2 Long term storage of cells

Approximately 1x10® cells were resuspended in 1 ml of freezing medium (Appendix 1) and slowly frozen in an alcohol bath (VWR) for 24 hours at -80°C. For long-term storage, cells were transferred to a liquid nitrogen dewer flask.

2.5.3 Resuscitation of cells from storage

Frozen aliquots of cells were removed from liquid nitrogen, defrosted quickly at 37°C and added to pre-warmed medium. The cells were then gently pelleted at 700 g to remove the DMSO containing freezing medium, and resuspended in fresh medium.

Chapter 2 - Materials and Methods

2.5.4 Analysis of cell viability

Cells in culture were analysed for viability by using the vital stain trypan blue, that only enters across the membranes of dead or non-viable cells. Cells which have been treated with the stain and are healthy appear round and retractile in contrast with dead cells which appear dark blue, non-refractile and often larger, when in suspension under a microscope. In order to count the number of viable or non-viable cells, adherent cells were first trypsinised and then resuspended in culture medium. Suspension cells were just resuspended to break up clusters of cells. Cells were diluted to an appropriate density, diluted 1:1 in 0.4% (w/v) Trypan blue (Sigma) and applied to the counting chamber of a haemocytometer. Viable cells were counted in at least two 1 mm^ squares of the haemocytometer, ensuring that more than 200 cells were counted to improve accuracy. Non-viable cells were also counted in the same areas and the percentage of viable cells could be calculated.

2.5.5 Lipofectamine transfection and luciferase assays

Lipofectamine reagent (GibcoBRL) was used to transfect mammalian cells with plasmid DNA. Careful optimisation of transfection conditions was essential to maximise transfection efficiency and lower toxicity. Conditions optimised included lipofectamine and DNA concentrations, cell number and time of exposure to DNA-liposome complexes.

2.5.5.1 Lipofectamine transfection

In a typical transfection reaction, cells were seeded at a density of 5 x lO'^ cells per well of an eight-well chamberslide and allowed to grow for 24 hours. The following solutions were then prepared for each transfection reaction: Solution A: 100 ng of plasmid DNA in 50 pi serum free medium and Solution B: 2 pi of Lipofectamine reagent in 50 pi of serum free medium. The two solutions were combined, mixed gently and incubated at 22°C for 30 minutes to allow DNA-liposome complexes to form. The cells were rinsed in serum-free medium and the DNA-liposome solution was added to the rinsed cells and incubated for 3 hours at 37°C. Following incubation, 100 pi of growth medium containing double the usual concentration of serum and antibiotics was added to each well. The cells were incubated for 24 hours and the medium replaced with fresh medium 24 hours after the start of transfection. The cells were then assayed for the gene of interest 24-72 hours later, depending on the cell type and plasmid vector used.

Chapter 2 - Materials and Methods

2.5.5.2 Dual luciferase reporter assay

1x10® CHO cells were plated on to twelve well plates and grown for 48 hours. Cells were transfected with 2 pi of lipofectamine and 300 ng of plasmid DNA for 15 hours in serum-free medium. Fresh medium containing serum and varying levels of gentamicin were added to the transfected cells for 24 hours prior to being assayed. Cells were lysed and luciferase activity was determined using the dual luciferase reporter assay (Promega, Southampton, UK) according to manufacturer’s protocols using a Berthold Orion luminometer (Berthold, UK).

2.5.6 Immunocytochemistry

2.5.6.1 Adherent cells lines

Cells were seeded onto 8 well chamber slides at a density of approximately 30,000 cells per well and allowed to adhere and proliferate for 1-2 days before processing. The cells were rinsed twice in PBS and fixed in 3.7% formaldehyde for 20 minutes followed by detergent permeabilisation in 0.05% Triton X-100 for 15 minutes. Non-specific binding was blocked by incubation with 3% bovine serum albumin, 10% normal donkey serum in PBS (blocking buffer) for 1 hour at 22°C. The use of primary antisera was carefully optimised to ensure accurate and reproducible staining. Primary antisera were added for 1 hour at 22°C at the titres stated in Table 2.1 and the primary antibodies were detected with cyanine-conjugated secondary antisera at the titres stated in Table 2.2, for 1 hour at 22°C. All antibody incubations were performed in blocking buffer with 3 x 5 minute washes with PBS carried out in between.

2.5.6.2 Suspension cell lines

Lymphoblastoid cells were washed with PBS and trypsinised for 5 minutes in 0.01% (w/v) trypsin and 1 mM EDTA to reduce the cells’ natural tendency to clump together. After further PBS washes, the cells were fixed and processed using the same protocol as adherent cells. All wash steps and antibody incubations were carried out in 1.5 ml eppendorf tubes, gently mixed end-over-end. Cells were carefully pelleted at 700 g to ensure no damage to the cells in a bench top centrifuge in between each step. Approximately 1x10^ stained lymphoblastoid cells in a volume of 100 pi of were fixed to glass slides by cytospin centrifugation for 5 minutes at 100 g, and analysed using a Zeiss LSM510 laser scanning confocal microscope.

Chapter 2 - Materials and Methods

2.5.7 Sucrose gradient centrifugation

2.5.7.1 Basic subcellular fractionation

Lymphoblastoid cells were washed twice in PBS, pelleted by centrifugation at 1000 g

and resuspended in cell breaking buffer containing mammalian protease inhibitors (Sigma). The cells were allowed to swell for 10 minutes then broken by passing them 30 times through a steel ball bearing cell homogeniser (HY Enterprises, Redwood City, CA). After breakage the cells were centrifuged at 1870 g and the supernatant fraction removed for further resolution on a sucrose gradient.

2.5.7.2 Sucrose gradient fractionation

The supernatant fraction from the basic subcellular fractionation was further separated on a well characterised sucrose gradient (Lewis et al., 1992; Chappie et al., 2000). A 14 ml 10-40% (w/v) sucrose gradient in 50 mM HEPES pH 7.2, 90 mM KCI was prepared with a 65% (w/v) sucrose cushion. A 1 ml supernatant fraction was applied to the gradient and centrifuged using a swing-out rotor at 78800 gmax for 16 hours at 4°C. For the detergent treated fractions, NP40 was added to the supernatant at a final concentration of 0.5%, 10 minutes prior to loading onto the gradient. After centrifugation, 1 ml fractions were collected from the bottom of the tube (65% sucrose) and analysed by western analysis.