Flow cytometry and cell sorting

Flow cytometry analyses involved analysis of the endogenous fluorescent protein and immunophenotypes of the cells. They were performed using FACSCalibur (Becton Dickinson), or FACSCanto II (Becton Dickinson) when it required a blue laser.

2.3.1.1. Endogenous fluorescent protein analysis

The transduced cells that have fluorescent protein marker could be measured using flow cytometry. A small number of the cells (as few as 105 _{cells) were put into a}

FACS tube, diluted to 4 ml using PBS, and centrifuged (450 g for 4 min) to prevent colour interference from the growing media. The cells were resuspended in PBS at a maximum concentration of 106 _{cells/ml and measured using appropriate fluorescent}

channels (Table 2-21).

2.3.1.2. Surface marker staining and analysis

A small number of cells (1 – 5 x 105 _{cells) were collected into FACS tubes, made up}

to 4 ml using PBS, and centrifuged at 450 g for 4 min. They were resuspended in 50 μl of 0.2% PBSA (0.2% BSA in PBS) containing the labelled antibodies (antibody volume was based on the manufacturer’s recommendation) and incubated for 20 min in the dark at room temperature. Subsequently, the samples were washed by adding 3.5 ml of 0.2% PBSA and centrifuged, to remove the untagged antibodies. The samples were resuspended in 500 μl of 0.2% PBSA and measured using the appropriate fluorescent channels (Table 2-21).

63  Fluorochrome Fluorescent channel

APC FL4 eGFP FL1 FITC FL1 PE FL2 PE-Cy7 FL3 PerCp-Cy5.5 FL3 RFP657 FL4 tdTomato FL2 Venus FL1

Table 2-21 Fluorescent channels for different fluorochromes on FACSCalibur. APC-Cy7 and

BV421 were analysed using FACSCanto II due to no available filters and laser in FACSCalibur. Also, the fluorescent channels have been named according to the fluorochromes in FACSCanto II, apart from eGFP and Venus, tdTomato, RFP657, and BV421 that were analysed using FITC, PE, APC, and Pacific Blue channels, respectively.

2.3.2. Cell sorting

Cell sorting techniques were used to isolate fluorescent protein expressing cells, labelled cells populations, or single cells. Three different instruments were used: FACSAria III (Becton Dickinson), Astrios EQ Cell Sorter (Beckman Coulter) and FACSAria Fusion Cell Sorter (Becton Dickinson). The FACSAria III was located in NICR and experiments were carried out by Hesta McNeill, while others are located with and performed by the Flow Cytometry Core Facility, Newcastle University. The sample labelling for haematopoietic sub-population sorting was carried out with assistance from Dr Paul Milne, who had previously optimised the antibody panel used.

2.3.2.1. Fluorescent protein expressing cells sorting

Before sorting, the population of fluorescent protein-expressing cells was evaluated (section 2.3.1), to have an estimate of the number of cells that would be collected. After that, the desired number of cells was centrifuged (300 g, 5 min) and

resuspended in sorting buffer at a concentration of 1 – 2 x 107 _{cells/ml. The cells}

were then passed through a 40 µm cell strainer to remove any clumped cells and transferred into a FACS tube to be sorted.

The cells were sorted and collected into a 1.5 ml microfuge tube, FACS tube, or 15 conical tube containing 500 μl, 1 ml, or 2 ml culture media, respectively, depending

on the number of cells to be collected. Afterwards, the cells were cultured under standard conditions.

2.3.2.2. Haematopoietic sub-population sorting

Haematopoetic sub-population analyses were performed on primary and primograft patient samples. The sorting preparation was assisted by Dr Paul Milne. Up to 107

cells were prepared, centrifuged, and resuspended in 100 µl sort buffer. The

antibodies were added and incubated in the dark at 4°C for 30 min. The antibodies to be used are listed in Table 2-22. After staining, the cells were washed, resuspended in sort buffer, and passed through a 40 µm cell strainer to remove any clumped cells and transfer into a FACS tube, and sorted.

The sorted cells were collected into 1.5 ml microfuge tubes containing 500 µl RF-10 media. After sorting, they were added with additional 500 µl media to dislodge any cells that may attach to the upper part of the microfuge containing no media, and continued with further applications.

Antibody Fluorochrome Astrios filter

CD56 FITC 488/530 CD34 PerCP-Cy5.5 488/695 CD117 PE 561/586 CD19 PE-CF594 561/620 CD38 PECy7 561/795 CD33 APC 642/671 CD90 A700 642/722 CD3 APC-Cy7 642/795 CD123 BV421 405/448 CD45RA BV510 405/530 CD10 BV650 405/671 HLA-DR BV786 405/795

Table 2-22 Antibodies panel for haematopoietic sub-populations sorting on Astrios

2.3.2.3. Single cells sorting

Single cell analysis was started with the isolation of the single cells into 96 well plates (Eppendorf twin.tec PCR Plate 96 full skirt). The cells were sorted to the plates based on the design in Figure 2-1 that includes forty-five single cell samples, two 0 cells as

65 

96 well plate. This design provides the plate with only 48 total samples to have a good time allocation for distribution of the whole genome amplification (WGA) reagents.

Figure 2-1 Plate design for single cell sorting. The design included two 0 cells as negative controls,

one bulk (300 – 1,000 cells) as a positive control, and forty-five single cell samples.

Before the sorting, the plates were filled with 3.5 µl PBS/well and sealed with tape pad (it is recommended to prepare extra plates per cell type). The cells were

prepared from as many as 105 _{cells. They were centrifuged and re-suspended in 500}

µl filtered 0.2% PBSA.

When the preparations were finished, the plate to be sorted was centrifuged at 2,000 rpm for 1 min. The sorting was performed by the Flow Cytometry Core Facility team using FACSAria Fusion Cell Sorter. During sorting, plates not actively on the cell sorter were kept on ice. Following the sorting, the plate was resealed with new tape pad and centrifuged at 2,000 rpm for 1 min to ensure single cells were not attached to the no-media surface of the well. Plates were then stored at -20°C until further application.

1 2 3 4 5 6 7 8 9 10 11 12

A 0 0 0 0 0 0 0 0 0 0 0 0

B 0 0 0 0 0 0 0 0 0 0 0 0

C 0bulk ctrl sample sample sample sample sample sample sample sample 0 (neg ctrl) 0

D 0 sample sample sample sample sample sample sample sample sample sample 0

E 0 sample sample sample sample sample sample sample sample sample sample 0

F 0 sample sample sample sample sample sample sample sample sample sample 0

G 0 sample sample sample sample sample sample sample 0 (neg ctrl) 0 0 0

2.4. Molecular biology methods