LOS INGRESOS Y EL FINANCIAMIENTO DEL ESTADO.

Materials

* RPM I 1640 containing L-glutamine Gibco BRL, Paisley, UK

* Foetal C alf Serum(FCS) Gibco BRL, Paisley, UK

(heat inactivated for 30mins)

* Penicillin/Streptomycin (Pen/Strep) Gibco BRL, Paisley, UK

* Bovine Serum Albumin (30%) Sigma, Poole, UK

* Ethidium bromide Sigma, Poole, UK

* Fluorescein-di-acetate Sigma, Poole, UK

* Trypan blue Sigma, Poole, UK

2.3 .1 C ell L ines

The following established cell lines were used:

HL-60 derived from a patient with acute promyelocytic leukaemia.

Collins et al, 1977

Method

All cell culture studies were performed in sterile plastic tissue culture flasks. All cell lines were maintained in suspension culture in RPMI 1640 supplemented with 10% FCS and 2% Pen/Strep in a humidified atmosphere containing 5% CO2 at 37°C and subcloned twice a week to a density of 0.6-1x1 0^ cells/ml to ensure that exponential growth was maintained. Cell number was determined manually by microscopic counting using an improved neubûu^e^counting chamber. Cell viability was measured using an ethidium bromide/fluorescein-di-acetate viability stain.

2.3 .2 C ell coun ting and viability

Cell number was determined manually by microscopic counting using an improved neubeauer counting chamber.

Cell viability was measured using an ethidium bromide/fluorescein-di-acetate viability stain. This was prepared by adding 4ml o f ethidium bromide (4mg/ml) to 10ml of fluorescein-di-acetate (0.1% solution) and this solution was made up to 1 litre with PB S. The solution was stored in the dark at 4°C until required.

Viability was assessed by mixing 100>Aof cell suspension with 100/t/l o f ethidium bromide/fluorescein-di-acetate stain. The percentage of viable cells was counted using a

fluorescent microscope. A minimum o f 500 cells were counted for each sample. Ethidium bromide selectively enters dead cells which then fluoresces an orange-red colour, whilst, fluorescein-di-acetate is taken up by viable cells which appears a green colour under UV light (Mills etal, 1983).

2.3.3 F reezin g and thaw ing o f cell lines

For freezing, cells were suspended at 5-10x10^ cells/ml in 10% dimethyl sulphoxide (Sigma, Poole, UK) and 20% FCS in RPM I medium. Aliquots o f 1ml were frozen overnight in cryotubes at -70°C and then transferred to liquid nitrogen for storage. Frozen cells were rapidly thawed in a 37®C waterbath and diluted by dropwise addition o f RPM I 1640 medium supplemented with 10% FCS and 2% Pen/Strep to 20ml. Cells were washed once, resuspended and counted before use.

2.3 .4 C y to sp in p rep a ra tio n s

Single cell suspensions were prepared at 1.0x10^ cells/ml and 100//U o f cell suspension was added to each prepared funnel with labelled slides and filter attached. The centrifuge (Cytospin 2, Shannon) was spun at 700 rpm for 10 minutes. Slides were removed, air dried and stained with May Grunwald Giemsa staining for 30 minutes

2.3 .5 T h y m o cy te Iso la tio n

Thymocytes were obtained from male Balb-C mice and aged 10-12 weeks, unless otherwise stated. Following cervical dislocation, the thymus was carefully dissected and contaminating connective tissue removed. The thymocytes were released from the capsule by gentle teasing apart the thymus in culture medium (RPMI 1640 medium, supplemented with 10% FCS and 2% Pen/Strep), debris was removed from the suspension via filtration through a plastic gauze. The cells were suspended at a concentration o f 1.0x10^ cells/ml in culture medium and incubated in 5ml polypropelene tubes at 37°C in 5% C O2. Cells were counted using a haemocytometer and viability determined by trypan blue exclusion. Cells o f a >95% purity were used for subsequent experimentation.

2.3.6 C ulture o f bone m arrow cells from m urine bone m arrow

Male C57bL/J mice aged 10-12 weeks were sacrificed by cervical dislocation. The femur was isolated and the haemopoietic cells were flushed from the bone cavity with 5mis of RPM I medium supplemented with 10% FCS and 2% Pen/Strep, using a 25G hypodermic needle. The isolated cells were washed x2 with 5mis o f the same medium and

then resuspended at a concentration of 1x10^ cells/ml. After 24h incubation at 31^C with the drugs o f interest, cells were pelleted and fixed in 70% ethanol for 60mins. After this time, cells were washed x3 with 3% BSA/PBS, a rat anti-mouse 1^ neutrophil antibody (Serotec, Oxford, UK) was added at a dilution of 1:4 and left on ice for 30mins. Cells were once again washed x3 with 3% BSA/PBS to get rid of any free antibody. The 2^ antibody, a F(ab')2 Rb anti-rat IgG(AFF.PUR): FITC (Serotec, Oxford, UK) was added at a dilution o f 1:50 and again left on ice for 30mins. After several washes, cells were resuspended with 0.25ml of 3% BSA/PBS, 0.25ml RNase, 1 mg/ml and 0.5ml propidium iodide, 50pg/m l. Cells were left in the dark at 4^C until analysis on the FACS for % of apoptosing cells as oulined in section 2.4.1

2.3.7 Isolation o f Peripheral blood progenitor cells

Patients with haematological malignancies were mobilised with either cyclophosphamide (1.5g/m^) and G-CSF (263 pg daily) or ESH AP chemotherapy (etoposide 40mg/m^ for 4d, cA-platin 25mg/m^ over 4d, cytarabine Ig h rfi over 4d and methylprednisolone 500mg daily) and G-CFS. CD34+ cells were then isolated from the leukopheresis product by one of two methods. In some cases the entire hai'vest product was positively selected using the Ceprate SC stem cell concentration system and the 12.8 biotinylated anti-CD34 antibody as previously described (Watts et al, 1996). Alternatively, a small aliquote o f the leukapheresis product was removed and the CD34-I- cells were positively selected using the Variomacs TM system (Miltenyi Biotech) as per manufactures instructions. Briefly, mononuclear cells were isolated by Ficoll-Flypaque deASity centrifugation. The cells from the interface were washed and incubated with lOOpl of multisort magnetic beads conjugated to the anti-CD34 monoclonal antibody Q-BEND/10 for 30 minutes on ice then selected on a variomacs column. Product purity was determined by APAAP (alkaline phosphatse anti-alkaline phosphatase) for the CD34 antigen on cytospins as previously described (Watts et al, 1996).