Flow Cytometry

Flow cytometry was used to analyse cell surface marker expression on transduced cell populations to examine the impact of retroviral expression on cell lineage. The technique was also used to investigate engraftment of injected cells in xenograft samples.

Flow cytometry was performed using several different BD machines and fluorochrome panels. The machines used were FACSCanto II, Fortessa X20 and for cell sorting FACSAria II. All markers used and corresponding paired lasers are displayed in section 2.2.10.1.

2.3.8.1 Flow Cytometry Compensation Set Up

The use of multiple flow markers facilitates the necessity of compensation alignment to minimise the expression of false positives or negatives due to the overlap of fluorochrome emission spectra. Compensation set up was performed using Anti-Mouse Ig, κ/Negative Control Compensation Particles (BD Bioscience).

Two drops of Anti-Mouse Ig, κ beads and two drops of negative control beads were mixed in a Falcon 5ml round bottomed polystyrene test tube for each fluorochrome to be compensated, excluding CD33 PE-Vio770 which was incompatible with the Ig, κ beads due to the composition of the stain as a λ chain antibody. CD33 compensation was set up with CD34+ cells cultured for at least two weeks in standard liquid media (Section 2.3.2.3). ~ 1 x 106 _{of cells of a} comparable morphology and marker expression of those to be tested were also stained with single antibodies, and were treated identically to the beads. An additional negative unstained cell control was prepared. Antibody concentrations for fluorochromes were ~0.20µg per 1 x 106 _{cells. Antibody stains were applied}

109

and beads/cells were incubated in the dark for 15 minutes at room temperature or for 30 minutes at 4°C. Samples were washed with 1ml of FACS Buffer and centrifuged at 400 x g for 5 minutes. Supernatant was discarded and the wash step was repeated and supernatant was discarded. Samples were then prepared for compensation set up on the FACS machine of choice.

Negative and single fluorochrome cell preparations were used to adjust machine photomultiplier tube (PMT) voltages to ensure all desired cells were analysed. PMTs are optical detectors, which record the presence of fluorescence, converting photons to electrical signals as stained cells pass through the relevant lasers. Adjusting the voltage to the PMTs determines the intensity of the PMT signal. After PMT voltage had been adjusted a compensation wizard program provided with the FACS Diva suite was opened and prepared to capture the single antibody stains comprising the multicolour FACS panel. The compensation particles were analysed and data captured, gates were created to capture the positively stained Ig, κ beads, and the negative beads. This was repeated for each fluorochrome in the particular multicolour panel. CD33 compensation was performed using stained CD34+ cells as positive controls and unstained CD34+ cells as negative controls. After each stain was captured the FACS Diva program performed automatic compensation on the multicolour panel based on captured data, although compensation could also be adjusted at a later date if necessary. The experiment was saved, ready for use with experimental cells.

2.3.8.2 Analysis of Transduction Efficiency

Transduced cells were analysed using flow cytometry. EGFP expression representing MSCV-IRES-EGFP vector transduction and Thy1 expression representing MSCV-IRES-Thy1 vector transduction, which was tagged to the PE fluorochrome. Both markers were compared against positive and negative controls.

2.3.8.3 Analysis of Cell Surface Phenotype

In vitro cells were analysed weekly, up to 1 x 106 _{cells were isolated and} aliquoted into individual pre-labelled 5ml polystyrene tubes. The cells were washed with 1ml of FACS Buffer and centrifuged at 400 x g for 5 minutes.

110

Supernatant was discarded and cells were stained with the relevant FACS panel (Supplementary Table 7.2, Supplementary Table 7.3) and 50µl of FACS Buffer. The cells were incubated in the dark for 15 minutes at room temperature or for 30 minutes at 4°C. Samples were washed with 1ml of FACS Buffer and centrifuged at 400 x g for 5 minutes. Supernatant was discarded and the wash step was repeated and supernatant was discarded. The samples were stored in the dark and used within 2 hours of preparation. Cells were run with a pre- prepared compensation set up (Section 2.3.8.1).

2.3.8.4 Thy1 Biotin-Streptavidin Labelling

Thy1 expressing cells were labelled with biotin conjugated CD90 antibody and stained with PE Streptavidin for visualisation using flow cytometry.

Cells were isolated and labelled with 0.5µl of biotin antibody in 20µl of FACS buffer per 200k-1 million cells, incubated for 30 minutes at 4ᵒC, washed with FACS buffer, and pelleted by centrifuging at 1200g for 5 minutes, supernatant was discarded. Cells were resuspended with 0.5µl of PE Streptavidin in 20µl of FACS buffer and incubated for 30 minutes at 4ᵒC, washed with FACS buffer, and pelleted by centrifuging at 1200g for 5 minutes. Supernatant was discarded and cells were analysed using flow cytometry.

2.3.8.5 Cell Sorting

Cells were treated identically to section 2.3.8.3, with the exception being that the amount of antibody used was raised to ~0.40µg per 1 x 106 _{cells. Cells were} sorted on the FACSAria II. Single colour controls were analysed prior to sorting to optimise PMT voltage and compensation settings. Sorted cells were resuspended in RLT buffer and stored at -20°C before use.

Cell Fixation and DAPI Staining

Cells grown in vitro were analysed weekly, up to 1 x 106 _{cells were isolated and} aliquoted into individual pre-labelled 5ml polystyrene tubes. Samples were washed with 1ml of FACS Buffer and centrifuged at 400 x g for 5 minutes, supernatant was discarded. Cells were then fixed using 2% Formaldehyde,

111

0.01% Triton X-100 solution. Cells were incubated at room temperature between 8-16 hours, cells were centrifuged at 400 x g for 5 minutes and supernatant was discarded. Samples were washed with 1ml of FACS Buffer and centrifuged at 400 x g for 5 minutes, supernatant was discarded. Samples were resuspended in 500µl of DAPI solution and were incubated in the dark at room temperature between 8-16 hours. Cells were centrifuged at 400 x g for 5 minutes, supernatant was discarded. The cells were then ready to be analysed on the flow cytometer. Cells were captured at a low flow rate for optimal results.