Plan de Acción Estratégico

4.2.1 Routine maintenance of cell lines

Cells were maintained in T75 Costar tissue-culture grade flasks in a 37°C humidified incubator with 5% C 0 2. Cultures were split when subconfluent, the harshness of the split ranging from 1:5 to 1:20 depending upon the cell line and experimental requirements. The media used to maintain each cell line are shown in table 4.2. To facilitate attachment of the PC 12 cells, flasks were coated with rat tail collagen according to the method of Green et al. (1987). All cell lines were split using trypsin and Versene. Briefly, the medium was removed from a flask by aspiration and replaced with 5m! Versene, prewarmed to 37°C. The Versene was left in contact with the cells for five minutes to chelate divalent cations, specifically calcium and

magnesium. The Versene was then removed by aspiration and replaced with 5ml 0.25% w/v trypsin, also prewarmed to 37°C. The trypsin was left in contact with the cells for up to 5 minutes and then the cells were removed from the surface of the flask by gentle agitation. 10ml complete medium, prewarmed to 37°C, was then added to the flask and the cell suspension collected into a 20ml Sterilin tube. The cells were pelleted by a 3 minute centrifugation at 1000 rpm and resuspended in 10ml fresh medium. 0.5ml - 2ml of the suspension was used to reseed a fresh flask containing

15ml medium prewarmed to 37°C. Cells which tended to clump together, such as the PC 12 (Geneva) and P19 cells, were removed from the plastic surface using a Costar cell scraper and a small volume of medium. The cells were then collected into a 20ml Sterilin tube and monodispersed by pipetting up and down a number of times.

M aterials an d M ethods: C hapter 4

Cell line Maintenance Medium

Ltk- DMEM + 10% FBS

HeLa DMEM + 10% FBS

U138MG DMEM + 10% FBS

U373MG DMEM + 10% FBS

Neuro 2A DMEM + 10% FBS, 1%NAA

NB4-1A3 Ham's F10 + 12.5% HS, 2.5% FBS

PC 12 (Geneva) RPM11650 + 10% HS, 5% FCS

PCD (Sheffield) DMEM + 10% FBS

P I9 (stem cells) DMEM + 10% FBS

NTERA-2 (stem cells) DMEM + 10% FBS

Table 4.2 - Maintenance media required for cell lines used in the present study. All media were also

supplemented with 2mM L-glutamine and 10 U m l"'/0.1 mg m l'1 penicillin/streplomycin.

Abbreviations: DMEM - Dulbecco's Modified Eagle's Medium, FBS - Foetal Bovine Serum, NAA - Nonessential Amino Acids, FIS - Horse Serum, FCS - Myoclone Foetal Calf Serum.

4.2.2 Storing cell lines

For long term storage, cells were dissociated and pelleted as described in the

preceding section. They were then dispersed in 10ml fresh medium, in order to dilute out the trypsin and ensure that all clumps were separated, and pelleted once again. The cells were then dispersed in 5ml freezing mix (90% v/v serum, 10% v/v DMSO) and divided into 1 ml aliquots in freezing vials. The serum in the freezing mix was made up to reflect the requirements of the proliferating cells, thus for Ltk- cells a mixture containing 90% v/v foetal bovine serum and 10% v/v DMSO was used whilst for PC 12 (Geneva) cells, a mixture containing 60% v/v horse serum, 30% v/v myoclone foetal calf serum and 10% v/v DMSO was used. The cells were

immediately transferred to a storage box at -70°C and left overnight. They were then stored indefinitely under liquid nitrogen.

4.2.3 Differentiation of PC12 cells using nerve growth factor

PC 12 cells were induced to differentiate with NGF-P from mouse submaxillary gland (Sigma, St. Louis MO, USA). PC 12 (Geneva) cells were differentiated with complete medium supplemented with 50ng mb1 NGF according to the method of Green et al. (1987). PC 12 (Sheffield) cells were differentiated according to the same method but using 1 OOng mb1 NGF (R Gibson, pres. comm.). Differentiated cells for transfection were seeded at a density o f 5 x 105 into collagen-coated Nunc 35mm plates.

M aterials a n d M ethods: Chapter 4

4.2.4 Differentiation of EC cells using retinoic acid

P19 mouse EC cells were induced to differentiate with retinoic acid according to the method of Rudnicki and McBurney (1987). Differentiated neuronal cells were selected by supplementing the growth medium with 5 pg mH cytosine arabinoside 48 hours after retinoic acid treatment was discontinued, thus inhibiting the growth of proliferative cells (Rudnicki and McBurney, 1987).

4.2.5 Transfection of eukaryotic cells - general strategy

Transfections were carried out in triplicate using Nunc 35mm 6-well plates. Each experiment was performed at least twice (lipofections) or three times (other transfection methods). Two independent preparations o f each construct were tested (plasmids were prepared using the double caesium chloride equilibrium centrifugation method described in section 4.1.1). In pilot experiments designed to optimise

transfection efficiency, cells were transfected with pSV-P-galactosidase, a plasmid which contains the E. coli lacZ gene under the control of the SV40 early promoter, and transfection efficiency was determined by soluble P-glactosidase assay. To analyse transcriptional regulation o f the rat NSE gene, constructs containing parts of the NSE 5' flanking region joined to the E. coli cat gene were transfected into a variety of neuronal and nonneruonal cell lines and the transcriptional activity of each construct was determined by CAT assay. As internal controls, pCAT-Control, a plasmid which contains the E. coli cat gene under the control of the SV40 early promoter, and pCAT-Basic, a plasmid which contains the E. coli cat gene but lacks a eukaryotic promoter, were transfected into parallel cultures as standards to compare promoter function. Each construct was cotransfected with an equal amount (by mass) of pSV-P-galactosidase to control for transfection efficiency across all constructs. The cotransfection control plasmid allowed the data from CAT assays to be normalised for transfection efficiency as shown in section 5.5.1.

4.2.6 Transfection using DEAE-dextran

DEAE-dextran mediated transfection was used successfully for Ltk- cells but was found unsatisfactory for other cell types. The method used was that of Selden (1987) and after careful optimisation, 2|ig DNA and 200pg DEAE-dextran (added as a 1 Omg

m l'1 solution i" TBS) made up in 1.5ml OptiMEM serum-reduced medium were used per 35mm well- Transfection was carried out at 50-60% confluence with a duration 4 hours followed hy 2 minute 10% v/v DMSO/PBS shock at room temperature. Chloroquine treatment’ which has been reported to increase transfection efficiency (Luthman and Magnusson, 1983) was not attempted.

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4.2.7 Transfect'011 usi"g calcium phosphate

Transfection by ca' c‘urn phosphate was carried out according to the modified procedure o f C ^en 3,1(1 Okayama (1987; 1988) as described in Sambrook et al. (1989), ppl6.39-16.40 Several batches o f 2x BES-buffered saline were prepared and tested for transfectlon efficiency using mouse Ltk- cells. The optimal buffer was stored and used 38 3 reference for the preparation o f further buffer stocks.

Coprecipitates vVere PrePared in 1ml volumes which were divided equally between triplicate wells- The optimal amount of DNA used per transfection was carefully optimised for e"ch individual cell line.

4.2.8 T ra n s fe c t'0" “ sing liposome formulations

Several différé"1 lip°sorne formulations have been used to transfect cells during this project (seeTable 4-3). Liposome-mediated transfection was carried out in the first instance according to the manufacturers' general guidelines and then carefully optimised for e"ch cell line by modulation o f the transfection parameters according to the m anufactures' instructions. The choice of liposome formulation itself appeared to be critical for the efficient transfection of some cell lines and a number of different products were compared wherever possible. The critical parameters for liposome mediated transfect*on were transfection duration, concentration of lipid, concentration o f DNA and the presence or absence of serum in the transfection medium. Table 4.4 shows the optirr>um parameters for the cell lines used in this project.

M aterials and M ethods: C hapter 4

Liposome formulation Manufacturer

DOTAP Boehringer Mannheim

Lipofectin Gibco BRL

LipofectAMINE Gibco BRL

Tfx-50 Promega

TransfectACE Gibco BRL

Transfectam Promega

Table 4.3: Liposome formulations used during this project and their manufacturers

Cell line Lipid / amount (pi) DNA (pg) Duration (hrs) Medium

Ltk- LipofectAMINE 5 2 5-8 OptiMEM

NB4-1A3 LipofectAMINE 5 1.5 5 OptiMEM

HeLa LipofectAMINE 6 2 8-10 OptiMEM

P19 (stem cells) LipofectAMINE 10 2 4 OptiMEM

PC 12 (-NGF) LipofectAMINE 6-10 2 5 OptiMEM

Table 4.4: Optimal conditions for liposome-mediated transfection of a number of cell lines. Amounts for lipid and DNA are totals per 35mm well with 1ml medium. LipofectAMINE is supplied at 2mg ml"*. OptiMEM is serum reduced medium developed for transfection (Gibco BRL). PCI2 cells require collagen coated flasks for attachment (see section 4.2.1).

4.2.9 Preparation of cell lysates

Transfected cells were scraped into microfuge tubes using a Costar disposable scraper, pelleted by 30 second pulse centrifugation and resuspended in 100pl 0.25M Tris.Cl (pH 7.5). The cells were lysed by three freeze-thaw cycles, and the debris pelleted by centrifugation at 13 000 x g for 10 minutes. 30pl of the cleared lysate was transferred to a fresh microfuge tube and assayed for p-galactosidase activity as described in section 4.2.10 whilst 50pl of the remaining lysate was transferred to a second fresh microfuge tube and assayed for CAT activity as described in section 4.2.11.

4.2.10 Soluble p-galactosidasc assay

Assays for p-galactosidase activity in mammalian cell extracts were carried out exactly as described in Sambrook el al. (1989), pp 16.66-16.67.

M aterials and M ethods: Chapter 4

4.2.11 CAT assay

CAT activity was assayed by thin layer chromatography, essentially as described by Gorman et al. (1982) although there were minor differences between the method used in this project and the published protocol. The CAT reaction mixture comprised 50pl cell lysate, 5pl 25pCi m l'1 D-threo-[l, 2-14C]-chloramphenicol, 5pl 66mg m l'1 acetyl coenzyme A and 40pl sterile redistilled water. Reactions were carried out for 1 hour at 37°C. Following chromatography, the plates were dried and exposed to X-ray film in an autoradiograph cassette without intensifier screen at room temperature.

Quantification was carried out by wrapping TLC plates in cling film and exposing them to a phosphorimager screen (Molecular Dynamics) for 4 hours. Phosphorimager analysis was facilitated by using the ImageQuant programme (Molecular Dynamics) according to manufacturer's instructions.