MATERIALS AND METHODS
2.11 Karyotype Analysis
Chromosomes of C57BL/6 PαS MSCs at P3 and P5 were analysed for the presence of any culture-‐induced abnormalities. The same culture was maintained between both time points, allowing us to identify whether any clonal growth of abnormal cells took place (Figure 2.4). This work was done in collaboration with Ms Mary Strachan, Dr Sara Dyer and Prof. Mike Griffiths at the West Midlands Regional Genetics Laboratory, Birmingham Women’s Hospital.
Figure 2.4 | Overview of PαS MSC karyotyping. PαS MSCs were grown in SM ± GFs. An aliquot of cells was taken for G-‐banding analysis at P3 and the same culture was maintained until P5 when another aliquot was taken. This allows us to detect any clonal expansion that could have taken place.
2.11.1 PαS MSC Harvesting
PαS MSCs were expanded as described previously in SM±GFs. When cultures reached 70% confluency, a 1:50 dilution of B-‐ONC (BRDU-‐overnight colcemid) was added and incubated overnight. Colcemid prevents spindle fibre formation and arrests dividing cells in metaphase.
Fresh colcemid was added the morning after to each flask and incubated for an hour. Samples were washed in PBS and harvested using TrypLE to yield a single cell suspension. Cell pellets were slowly resuspended in a hypotonic 0.025M KCL solution and incubated at 37˚C for 20 minutes. This solution causes the cell nuclei to swell in size to prevent the chromosomes from overlapping each other. After incubation, samples were spun at 1500rpm for 5 minutes and the supernatant discarded. The pellet was resuspended in the residual volume that remained and flicked gently to ensure a single-‐cell suspension. Samples were then slowly fixed in Carnoy’s fixative (1 part glacial acetic acid to 3 parts methanol). Samples were then spun at 1500rpm for 5 minutes and further fixative added in a drop-‐by-‐ drop basis. The supernatant was discarded after centrifugation and sample tubes stored at 4°C until slide making and Giemsa banding (G-‐banding).
2.11.2 Chromosome Spreads
The fixed cell suspensions were used to make slides for cytogenetic examination. Fixed cell samples were aspirated in a pipette and held over the middle of each slide from a height of approximately 50cm. One drop was allowed to fall and hit the slide, whereupon the nuclei would burst and the chromosomes would spread across the slide. Slides were checked to ensure minimal overlapping chromosomes and the cell density altered if necessary by adding more fixative. Slides were then ‘aged’ overnight at 60°C prior to G-‐banding.
2.11.3 Giemsa Banding, Imaging and Analysis
Leishman's stain (belonging to the same family of stains as Giemsa) was used in this study to mark chromosomes for cytogenetic analysis. The banding pattern observed with Leishman’s stain is virtually identical to Giemsa (Moore and Best, 2001). As such, both stains can be interchangeably used for analysis.
‘Aged’ slides were allowed to cool to room temperature and rinsed gently in tap water. Slides were then incubated in 0.25% trypsin for 90 seconds and then rinsed in saline solution. Slides were incubated in Leishman’s stain for 2 minutes before being rinsed in excess saline. Excess saline was removed using blotting paper and the slides were left on a hot plate at 60°C to air-‐dry before coverslipping.
Each slide was examined using a standard light microscope and images taken when a metaphase spread contains all 40 non-‐overlapping mouse chromosomes in the same optical field was observed. Individual chromosomes were electronically cut and arranged to produce a karyogram where sister chromatids are arranged in order. A total of 60 metaphase spreads per culture condition at P3 and P5 were analysed by Ms Mary Strachan for the presence of any karyotypic abnormalities. These findings were independently verified by Dr Sara Dyer before the final karyotype was reported.
2.12 Immunohistochemistry
2.12.1 Formalin-‐fixed Paraffin-‐embedded (FFPE) Tissue
FFPE tissue blocks were cut at 5µM thickness and fixed onto microscope slides (X-‐tra Adhesive, Leica). Slides were dewaxed and rehydrated by passing through xylene (3x 2 minutes each), IMS (2x 2 minutes each) and water (1x 2 minute wash). Antigen retrieval was performed using a high pH buffer (Vector Laboratories, Peterborough, UK). The buffer was pre-‐warmed in a microwave for 5 minutes at high power before slides were added. It was then heated for a further 15 minutes before being left to stand for 10 minutes. The buffer was cooled by the gentle addition of cold tap water until it reached room temperature.
Slides were then dried and sections were encircled using a wax pen. Endogenous peroxidase activity was blocked by incubating sections in a pre-‐diluted peroxidase blocking solution (Dako, Ely, UK) for 40 minutes. Slides were then washed in Tris-‐buffered saline + 0.1% Tween20 (TBS-‐T; 2x 5 minutes each) before blocking in 1x casein buffer (Vector Laboratories) for 60 minutes. Primary antibodies were diluted in TBS at the appropriate dilutions (Table 2.6) and added directly the after casein blocking stage. Slides were incubated with primary antibody at room temperature for 60 minutes with gentle rocking. Unbound antibody was removed by two further TBS-‐T washes (5 minutes each). Sections were then incubated with pre-‐diluted horseradish peroxidase (HRP)-‐conjugated secondary antibody (Vector Laboratories) for 30 minutes at room temperature with gentle rocking. Unbound secondary antibody was removed in three TBS-‐T washes (5 minutes each) before chromogenic visualisation.
3,3'-‐Diaminobenzidine (DAB; AbD Serotec, Kidlington, UK) was prepared according to manufacturer’s instructions and incubated with sections until brown staining was visible (typically around 30-‐120 seconds). Sections with an isotype-‐matched control (IMC) primary antibody were run at the same time to check for non-‐specific binding. Excess substrate was washed off in tap water for 5 minutes to stop the chromogenic reaction. Sections were then counterstained in Mayer’s haematoxylin and “blued” for 150 seconds in tap water. Slides were then dehydrated by passing through IMS twice and xylene three times before mounting coverslips using DPX. DPX was allowed to set overnight before imaging on a conventional light microscope (Carl Zeiss AG).
2.12.2 Snap-‐Frozen Tissue
In some cases, tissue was snap-‐frozen in liquid nitrogen and embedded in OCT compound (Sakura Tissue-‐Tek). 7µm sections were cut, mounted on pre-‐coated glass slides (Thermo Scientific), and fixed in acetone for 10 minutes. Excess acetone was removed in two washes of TBS-‐T (5 minutes each). Endogenous peroxidases were blocked by incubating slides for 15 minutes in a pre-‐diluted peroxidase blocking solution (Dako). Sections were then blocked in 1x casein buffer (Vector Laboratories) for 30 minutes before primary antibody (Table 2.6) was added. Sections were left to incubate at room temperature for 60 minutes with gentle rocking. Unbound primary antibody was removed in two washes of TBS-‐T (5 minutes each) and HRP-‐conjugated secondary antibody (Vector Laboratories) was added for 30 minutes at room temperature with gentle rocking. Sections were then washed, stained with DAB,