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The Chinese hamster ovary (CHO) CHO-K1 cell line, a proline-deficient

subclone from the original CHO line (which was first isolated by Puck et al.

(1958)), was used in experiments throughout this thesis. The CHO-K1 cell line

exhibits epithelial morphology and grows in an adherent monolayer. They

were maintained in Dulbecco’s modified Eagle’s medium nutrient mixture F12

(DMEM/F12) medium supplemented with 10 % foetal calf serum (FCS) and 2

mM L-glutamine (=growth medium) in cell culture incubators maintaining a

humidified atmosphere of 5 % CO2/ 95 % air. All cell culture techniques were

carried out in class II laminar flow cell culture hoods. All cell culture medium

and solutions used were pre-warmed in a 37 °C water bath before use to

passaging of cells into cell culture flasks and seeding of cells into plates for

experimentation, the cells were returned to the cell culture incubator (37 °C,

5 % CO2/95 % air atmosphere) to allow cells to adhere and grow until used for

further passaging or experimentation.

Passaging of cells

The cells used in this study were generally maintained in 75 cm2tissue culture

treated flasks (T75s) and grown to confluence. For passaging, the growth

medium was removed from the flask and cells were washed with 5-10 mL

phosphate buffered saline (PBS) to remove any remaining serum off the cells

and the flask. Cells were lifted off the bottom of the flask by incubation in 1

mL 1x trypsin-EDTA at 37 °C in a cell culture incubator. Trypsin is a serine

protease and is used here to hydrolyse proteins that facilitate the adherence

of the cells to the cell culture dish. After 2-3 minutes the cells were loosened

and easily dislodged by the addition of 5 mL growth medium, which also

prohibited any further action of trypsin on the cells. The cells were then

centrifuged at 1000 rpm for 5 minutes and the supernatant discarded. The

cell pellet was then resuspended in 10 mL growth medium and the

appropriate amount was transferred into a new T75 flask. Cells were routinely

passaged at a 1:10-1:20 dilution once a week. Multiple flasks were gained

from one T75 to keep the passage number low and flasks were used for

experiments the next week.

96-well plates

Cells were seeded into 96-well plates for CRE-mediated SPAP transcription,

ImageXpress (IX) Ultra confocal plate reader and PHERAstar FS plate reader

experiments. The cells from one T75 flask were detached from the bottom of

the flask as described above (see above Passaging of cells). The cell pellet was

then resuspended in 10 mL growth medium and 2 mL of this was then

transferred to a further 8 mL of growth medium (to make up 10 mL in total,

i.e. a 1:5 dilution). Of that, 100 µL were transferred into each well of a 96-well

plate (0.3 cm2 surface area per well). The same dilution (1:5) was used for

CRE-mediated SPAP transcription, IX Ultra confocal and PHERAstar FS plate

reader experiments, however, cells for SPAP gene reporter experiments were

plated out 48 hours prior to experimentation and cells for IX Ultra confocal

and PHERAstar FS plate reader experiments were seeded 24 hours prior to

experimentation. This was done to allow cells for SPAP gene reporter

experiments to be serum starved 24 hours prior to experimentation.

24-well plates

The cells were prepared into a 10 mL cell resuspension as described above for

96-well plates. The same dilution (1:5) was prepared and 500 µL of a 10 mL

cell suspension was added to each well of a 24-well cell culture plate (2cm2

surface area per well) for [3H]cAMP accumulation experiments. Cells seeded

into these plates were used for experimentation the following day.

6-well cell culture plates were used to seed cells to be used in confocal

perfusion experiments. A circular (3.2 cm in diameter) glass coverslip was

placed into each well of a 6-well plate. Cells of one T75 flask were lifted off

the bottom of the flask as described above (see above Passaging of cells). The

cell pellet was then resuspended in 6 mL of growth medium, of which 2 mL

(for a 1:3 dilution) were transferred to a further 10 mL of growth medium (for

a total of 12 mL). 2 mL of this cell suspension were then transferred onto the

glass coverslips in each well of a 6-well plate (9.6 cm2surface area per well).

The seeded cells were used in experiments the following day.

8-well glass chambers

Cells were prepared into a 10 mL resuspension as described above for 96-well

plates and seeded into 8-well Labtek borosilicate chambered-coverglass

plates (Nalgene Nunc International, Fisher Scientific, Loughborough, UK) for

confocal microscopy experiments. From the 10 mL cell suspension, 1 mL was

transferred into a further 19 mL of growth medium (1:20 dilution). From this

cell suspension, 400 µL were added to each well of an 8-well plate. The cells

were used in confocal imaging experiments two days after seeding.

Long term storage and thawing of cells

Foetal calf serum was supplemented with 10 % (v/v) DMSO (to constitute the

“freezing medium”) in the cell culture hood and subsequently sterilised by

filtration using a 10 mL syringe and a 0.2 µm sterile filter. The cryoprotective

freezing process, thus improving cell viability and recovery after thawing. Cells

to be frozen were detached from the bottom of a T75 flask and spun as

described above (see Passaging cells). The cell pellet was then resuspended in

2 mL of freezing medium by careful trituration before aliquoting 1 mL of the

cell suspension into a 2 mL labelled cryogenic tube. Thus, one T75 flask of

confluent cells generated two aliquots of cells in freezing medium. The tightly

capped cryogenic tubes were then put into an isopropanol-filled cryogenic

freezing container that allows controlled and gradual freezing of the cells at a

rate of 1 °C per minute in the -80 °C freezer. After 24 hours, the cryogenic

tubes were then placed into liquid nitrogen (-176 °C) for long term storage.

Cells in cryogenic tubes were taken out of liquid nitrogen storage and the

tubes were thawed in a 37 °C water bath (for circa 2 minutes). The cells were

then immediately placed into a T75 flask containing 20 mL pre-warmed

growth media. Working as quickly as possible reduces the risk of prolonged

stress for the cells and aids the survival and recovery of a larger proportion of

cells. The cells in the T75 flask were placed into the cell culture incubator at

37 °C and in a 5 % CO2/95 % air atmosphere. The following day, the growth

medium was replaced by fresh growth medium to remove any cell debris

from cells that had died during the thawing process. Cells were grown to

confluence and passaged once before being used for experimentation.