Toxicidad del Mercurio

2.2.13. Maintenance and Differentiation of Cell Lines

ND7 cells were grown in FGM consisting of L I5 (Liebovitz) medium (Gibco BRL with L-glutamine) supplemented with 10% v/v FCS, 5% v/v sodium bicarbonate, 0.35% w/v glucose and 2mM L-glutamine lOOx (Gibco BRL). DMEM (Gibco BRL with O .llg/L sodium pyruvate) supplemented with 10% v/v foetal calf serum (Gibco BRL, mycoplasma and virus screened) was used for transfections. N18Tg2 cells were grown in RPMI 1640 medium (Gibco BRL with L-glutamine) supplemented with 10% v/v FCS and 2.6% sodium bicarbonate.

To induce the ND7 cells to cease dividing and undergo morphological differentiation (Wheatly et a l, 1992) they were transferred to serum free medium, (SFM), consisting of a 1:1 mix of DMEM and Nutrient mix Ham's F12 (Gibco BRL with L-glutamine) supplemented with 5^ig/ml human transferrin (Gibco BRL), 250ng/ml bovine insulin (Gibco BRL) and 30nM sodium selenite (Gibco BRL).

N18Tg2 were induced to differentiate in RPMI 1640 medium supplemented with 0.5% newborn calf serum and 2.5mM sodium butyrate. Cells were typically passaged every 3-4 days and not used after 30 passages. Cells were detached from flasks by washing with ImM EDTA in BBSS, without Ca^+ and Mg^+ and without phenol red (Gibco BRL).

For long term cold storage cells were washed with HBSS/EDTA for 5 minutes, resuspended in 10ml of the standard medium and spun at l,000rpm for 5 minutes in an

lEC Centra 4R benchtop centrifuge. Approximately 1 x 1 0 ^ cells were resuspended in 500)il of culture medium and added to an equal volume of 15% DMSO in FCS (Gibco) before freezing overnight in cryostat tubes at -70°C in dry ice. Tubes were then transferred to liquid nitrogen. Cells were thawed rapidly, resuspended and spun as above and again resuspended in 5ml of medium and returned to small tissue culture flasks.

2.2.14. Selection of Stable Cell Lines

Cells were transfected as in 2.2.6. Following transfection cells were grown for 48 hrs in culture medium followed by transfer to conditioned medium supplemented with 0418 sulphate (Genetecin, Gibco BRL) or Hygromycin (Calbiochem) until only colonies resistant to G418 sulphate remained. Conditioned medium was prepared by removal from sub-confluent cells, filter sterilization and supplementation with 50% of the standard concentration of foetal calf serum. This medium was replaced every two days. ND7 cells were selected in 800^g/ml Genetecin, only cells that expressed the neomycin gene would surivive as this is the gene that confers resistance to G418 (Geneticin). Control, non-transfected cells grown in parallel and subjected to selection showed 100% cell death under these conditions. Isolated resistant colonies were picked from 6 well plates and grown in 96 well plates. When confluent cells were serially diluted in 24 well plates in 500^1 of conditioned medium containing Geneticin. Resistant colonies were picked and expanded for assay by Western blotting of protein extracts.

2.2.15. V iability Assays

A number of different approaches were taken in order to quantify cell death in cultures.

i). Trypan Blue Exclusion Assay

Cells were aspirated and spun for 10 minutes at 1250g and 4°C. The cell pellet was resuspended in 50^1 of medium and an equal volume of 0.04% Trypan Blue (Gurr, microscopy materials, BDH) in PBS was added. The cells were incubated at room temperature for 5 minutes and the proportion of cells able to exclude trypan blue counted in a haemocytometer counting chamber (Weber Scientific International Ltd., UK)(Gorman 1985).

it). Acridine Orange/Ethidium Bromide Staining

Rat neonatal (DRG) were seeded in Lab-Tek, 8 chamber permanox slides (Nunc, Inc.). Following treatment, the culture medium was removed and cells were incubated at room temperature for 5 minutes in 50|ig/ml ethidium bromide (Sigma) and Sp-g/ml acridine orange (Sigma) in PBS.

Cover slips were stuck on with nail varnish (Rimmel) and cells were visualized under UV light in a Nikon Diaphot microscpe using x40 objective.

iii). Cytotoxicity/Cell Proliferation Assay

Measurements of cell viability and proliferation were carried out using CellTiter

96™ Non-Radioactive Cell Proliferation/Cytotoxicity Assay (Promega) (Mosmann 1983; Tada et al.; 1986; Riss 1991).

iv). Giemsa Staining

Cells were cultured in six-well plates for the indicated period of time in serum-free medium either alone or with the addition of RA or cAMP. The cells were aspirated and centrifuged with 50ul of 20% bovine serum albumin solution onto microscope slides using a Shandon cytospin (500rpm, 5 minutes, low acceleration). After air-drying, the cells were fixed in ice-cold methanol for 2 minutes and stained with Giemsa stain: 5% Giemsa stain (Mercia diagnostics) in Sorensen's buffer pH 6.8 (Mercia diagnostics), with 0.3% anhydrous disodium hydrogen phosphate and 0.06% anhydrous potassium dihydrogen phosphate.

2.2.16. Cell Cycle Analysis using Flow Cytometric Analysis

The method used for distinguishing between the different phases of the cell cycle using flow cytometric analysis has been previously described (Burke et al.;

Cells were fixed in 70% ethanol at -2(PC, spun at 12,500 rpm for 5 minutes, washed, and resuspended in PBS containing 1 mg/ml RNase A (Sigma), 20^g/ml propidium iodide (PI; Sigma) and 5^g/ml fluorescein isothiocyanate (FITC; Sigma).

2.2.17 Heat Shock and Thermotolerance Experiments

Unless stated otherwise cells were heat shocked at 42°C for 30 minutes, using

a CO2 humidified incubator and medium that had been pre-heated to 42°C.

Typically cells were allowed to recover at 37°C for 150 minutes. To determine the effect of heat shock protein levels on thermotolerance at 42°C and 48°C, 200 cells were seeded onto 6 well plates (Nunc, Inc.) which were exposed to various periods at 42°C or 48°C. Cells were returned to 37°C and allowed to grow so that the number of seeded cells able to form colonies could be assessed.

2.2.18 Neonatal dorsal root ganglia cultures

Two day old Wistar rats were dissected aseptically to remove spinal gahlia. /■' Cultures were prepared by digesting ganglia with 0.3% collagenase (Boehringer Mannheim) for 60-90 minutes until ganglia could be mechanically dissociated through a 1ml Gilson tip. Cells were spun at 500g, plated onto 8 well Permanox tissue culture chamber slides (Lab-Tek Nunc, Inc.) coated with poly-L-lysine at a density of 1.6 x 10^ cells per well, and grown in F14 medium with 4% Ultraser G (Gibco BRL) and lOOmg/ml streptomycin and lOOU/ml penicillin plus lO^iM cytosine arabinoside. When appropiate, 2.5S NGF (Promega) was added to the cultures to a final concentration of 50ng/ml. Anti-NGF antibody was used at a dilution sufficinet to absorb 200ng/ml of NGF. Anti-ras antibody was used at the same dilution as a control

CHAPTER 3

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