2.2.2.1 Peripheral lymphocyte culture.
A synchronised culture method was used to produce extended chromosome preparations from peripheral lymphocytes o f controls, patients and their partners. Whole blood in sterile lithium heparinised tubes was cultured as soon as possible after collection. In a 50ml tissue culture flask, 1ml o f blood was added to a 20ml volume o f Iscoves modified Dulbecco's medium warmed to 37°C supplemented with 10% fetal calf serum, 1% glutamine, penicillin, streptomycin (GPS) and 1% o f the mitogen phytohaemoglutinin (PHA) under aseptic conditions (1A.1). The culture was incubated at 37°C for 48 hours with agitation twice daily to resuspend the cells. After this time 300pg/ml thymidine was added to block cell growth and incubation was continued for a further 18 hours before the block was removed by the addition o f 3pg/ml 2-deoxycytidine, allowing synchronisation o f a large proportion o f cells in the early stages o f mitosis. After incubation o f the culture for four hours O.lpg/ml colcemid was added to disrupt the mitotic spindle causing metaphase arrest. After 20 minutes o f colcemid incubation the culture was decanted into two 10ml centrifuge tubes and harvesting was commenced.
2.2.2.2 Harvest of cultured cells.
leaving approximately 1ml o f medium to resuspend the pellet. Hypotonic solution (0.075M KCl) pre-warmed to 37“C was added gently to prevent cell rupture and tubes were incubated for 15 minutes at 37°C. After this time the cells were spun down again at 6,000g for 5 minutes, the supernatant removed and the pellet resuspended in 1ml of residual medium. Freshly prepared fixative (3 parts methanol to 1 part glacial acetic acid) was then added drop-wise (approximately lOOpl) Avith a Pasteur pipette whilst the cells were constantly agitated by tapping the tube vigorously, until 5-10ml of fixative had been added. The cells were spun down as before, the supernatant removed and the pellet resuspended before the addition o f 5-10ml o f fresh fixative. This fixation step was repeated once more before storing cell suspensions at -20°C.
2.2.2.3 Cultured prenatal samples.
Surplus cultured prenatal samples including amniocytes, chorionic villi, and fibroblasts were obtained courtesy o f the Clinical Cytogenetics Unit, UGH. All were supplied as cell suspensions in 3:1 methanol:glacial acetic acid fixative.
2.2 2.4 Slide preparation I; Cell suspensions in fixative.
Slides were cleaned in cold methanol, 0.5% concentrated HCl and dried with a lint-free cloth. Prior to slide preparation fixed cell suspensions o f peripheral lymphocytes, fibroblasts, amniocytes or chorionic villi were centrifuged at 6,000g for 5 minutes, the supernatant was removed and the cell pellet was resuspended in fresh fixative (3:1 methanohacetic acid) until an appropriate cell density was reached. One drop (approximately lOOpl) o f suspension was dropped onto a clean dry slide and allowed to air-dry, before flooding with more fixative for 10 seconds and air-drying again. The slide was then flooded with 70% glacial acetic acid for 10 seconds to remove excess cytoplasm and air-dried. Cell density and chromosome spreading were checked under phase contrast microscopy and if necessary the process was repeated after adjusting the amount o f fixative added. Slides suitable for analysis with well spread metaphase chromosomes and minimal cytoplasm were dehydrated through an ethanol series (70%, 90%, 100% 5 minutes each), air-dried and stored covered at 4°C.
2.2.2.5 Slide preparation II; Blastomeres.
Slides were prepared o f embryonic nuclei from whole day three post insemination embryos and biopsied blastomeres using the method developed by Harper et aL, (1994) and Coonen et al., (1994a). Adhesive coated slides are used in this air-drying method to ensure cells remain attached to the slide until lysis and air- drying are complete. These were prepared by immersing clean slides in a coplin jar of tissue adhesive solution (0.01% Poly-L-Lysine in deionised water) for 5 minutes at room temperature before air-drying overnight, after which storage was at 4°C. The embryo or biopsied blastomere was washed in phosphate buffered saline (PBS) for 2 minutes to remove traces o f medium or oil before transfer to a small drop (approximately 10-20pl) o f spreading solution (0.0IN HCl, 0.1% Tween 20) on a Poly-L-Lysine coated slide, the position marked on the underside beforehand with a diamond pen. The slide was then transferred to an inverted microscope allowing the embryo to be constantly observed whilst a microcapillary primed with spreading solution was used to gently agitate the spreading solution drop. This was continued until the zona pellucida had dissolved, blastomeres lysed and all nuclei were clear o f cytoplasm. Slides were then air-dried, washed in PBS for 5 minutes and dehydrated through an ethanol series (70%, 90%, 100% 5 minutes each). Slides were stored covered at room temperature for a maximum o f two weeks prior to analysis.
2.2.2.6 Slide preparation III; Unfertilised oocytes.
Cytogenetic preparation o f unfertilised oocytes was carried out 48 hours post insemination to allow any late fertilised oocytes to be included for diagnosis. Chromosome spreading was by a gradual fixation method modified from Tarkowskis’ protocol (Tarkowski, 1966). Under an inverted microscope the oocyte was transferred using a microcapillary from culture medium to a petri dish containing 1% sodium citrate hypotonic solution and incubated for 6 minutes. The oocyte was then transferred to a small droplet (approximately 2pi) o f fixative I (5:4:1 bidistilled water:methanol:acetic acid) on a slide, the position marked on the underside with a diamond pen and allowed to dry. Fixation was concluded by the sequential addition of five 20pl drops o f fixative II (3:1 methanohacetic acid) using a pipette, with each one added just prior to drying o f the previous drop. Slides were then dehydrated
through an ethanol series (70%, 90%, 100% 5 minutes each), air-dried and stored at 4°C. Before FISH analysis all slides were stained with DAPI to distinguish analysable metaphases.