Introduction

2 .2 Cells, culture conditions and m aterials 2 .3 X-irradiation

2 .4 M utation assay

2.4.1 General mutation assay

2.4.2 Calculation of induced mutation frequencies 2.4.3 liability assay

2 .5 Survival assay

2 .6 Fixing and staining colonies 2 .7 Isolating m utant colonies 2 .8 Freezing cells for storage

- -- - - - .. ; ..

M aterials and m eth od s/22 2.1 Introduction

T his ch ap ter describes th e general m aterial and m ethods w hich w ere routinely u sed d u rin g th e course of th is project. T his includes cells, cu ltu re conditions an d the basic m utation assay used to isolate m u ta tio n s a t the autosom ally located tk locus in h am ster cells. The b asic m u ta tio n a ssa y u se d h a s b een described in general term s, how ever an y additional m odifications in any p a rt of the assay e.g th e exposure of cells to th e poten tial m utagen will be described in th e m aterials an d m ethod section of the respective chapter.

2 .2 C ells, culture conditions and m aterials

D uring the co u rse of th is project, all cell lines used, C hinese h a m s te r ovary (CHO KI), X -ray-sensitive m u ta n t cell line (xrs 5), C hinese h a m ste r (V79) cell line w ere routinely grow n a s m onolayers in 75 cm2 tissu e cu ltu re grade plastic flasks (Sterlin) an d m aintained in exponential grow th in 10 m l E agles M inim um E ssen tial M edium (MEM) su pplem ented w ith n o n -essen tial am ino acids an d 10% (v/v) fetal calf seru m (PCS). F lasks were gassed w ith 5% CO2 and incubated a t 37°C. Cells w ere p a ssag ed tw ice a w eek to m ain ta in cells in exponential grow th in all experim ents u n less otherw ise m entioned.

For p assag in g , m edium w as rem oved an d cells d etach ed from th e j su rface by trypsinization u sin g a trypsin/E D T A solution. Cells w ere J rin sed twice w ith 3 m l of trypsin/E D T A solution an d incubated for 6 | m in u te s a t 37°C. After th is period, cells w ere found to be detached

from th e surface of flask (seen as ro u n d ed free floating cells w hen view ed u n d e r a n in v erted m icroscope). To each flask, 5 m l of M EM /FCS w as added an d the su sp en sio n pipetted two to three tim es to give rise to a single cell su sp e n sio n . To d eterm in e th e cell co ncentration, 100|xl of su sp en sio n w as m ixed w ith 9.9 ml of iso to n so lu tio n an d co u n ted in a co u lter c o u n te r (m odel D) u sin g th e follow ing settings: T hreshold= 20; A ttenuation= 8; A perture c u rre n ts 0.017. The com position of routinely u sed solutions is described below:

Materials and m ethods/23

(a) Eagles m inim um essential m edium (MEM)

100 m is M inimum essential m edium ( xlO concentrate Gibco) 10 m is Penicillin + Streptom ycin,

10 mis G lutam ine

10 m is N on-essential m edium (Gibco) 30 m is Sodium bicarbonate

840 m is double-distilled w ater (b) Trypsin/E D T A

T rypsin (Difco) 0.05 % w ith 0.7 mM EDTA (BDH) in P h o sp h ate Buffered Saline.

(c) Trifluorothym idine supplem ented m edium (TFT/MEM)

C rystalline trifluorothym idine (trifuorothymine deoxyriboside. Sigma) w as w eighed an d dissolved in distilled w ater followed by filtratio n th ro u g h a m illipore filter (0.22 jiM, Flow laboratories). To give th e final w orking co n cen tratio n of 3pg/m l, the sto ck w as d ilu te d in M EM /FCS. All TFT supplem ented solutions w ere p ro tec te d from flourescent light.

(d) H A T-supplem ented m edium (HAT/MEM)

HAT m edia supplem ent (50x concentrate, Flow Lab.) w as diluted in M EM /FC S to give Ix co n cen trate solu tio n w ith th e follow ing co m position: h y p o x a n th in e (lOOjxM), am in o p terin (0.4pM ) a n d thym idine (16 |iM).

(e) 6-thioguanine supplem ented m edium

A stock solution of 6-thioguanine (2-am ino-6-m ercaptopurine, Sigma) w as prepared by dissolving in HESS followed by m illipore filtration (Flow Lab.). The final working concentration of Ijiig/ml w as obtained by subsequently dissolving in MEM/FCS.

(f) H ank's Balanced Salt Solution (HESS).

0.14M NaCl (8.0g/l), 5.4m M KCl (0.4g/l), 0.34m M N a2H PO4.2H2 0 (0.06g/l), 0.44m M KH2PO4 (0.06g/l), 6mM MgSO4.7H2 0 (1.5g/l), 5.6mM D-Glucose (lg/1), 4.2mM NaHCOa (0.35g/l). (All BDH).

Materials and methods/24

(g) H ank's Balanced Salt Solution (modified) + BSA,

HBSS (see above) + 1 % (lOg/1) Bovine Serum A lbum in (Fraction V). (BDH). (designated - HBSS/BSA)

(i) Fetal ca lf serum (FCS)

For th e grow th of cell cultures and m utageneisis experim ents, norm al m edium (MEM) supplem ented w ith 10% fetal calf serum w as used. All b atch es of FCS were tested to give a high plating efficiency and a low sp o n tan eo u s induced m utation frequency before using a b atch for th e m utation assay (Source of FCS: N orthum bria Biological Ltd)