FLOW CYTOMETRY

Flow assisted cell cytometry was used to analyse protein expression following various treatments.

2.4.1 Cell preparation and fixation

Following appropriate treatment, cells were trypsinised and counted before centrifugation at 1000xg for 3minutes. Cells were treated by fixation buffer (BD

Biosciences, UK) for 15minutes at room temperature and then washed in 5ml PBS with

0.1% (v/v) Triton X-100 (PBS/Triton). Cells were suspended at 1x106 _{cells/ml in}

2.4.2 Immunofluorescent staining

Cells were then allowed to pellet at room temperature and then the supernatant were carefully collected and discarded. Cells were then incubated with primary antibody solution (made in blocking buffer) for 1hour at room temperature (Table 2-11). Cells were allowed to pellet, and supernatant was collected and discarded followed by washing three times in PBS/Triton for 5minutes each time. Cells were then incubated in secondary antibody solution, made in blocking buffer, for 1hour at room temperature (Table 2-11). This was followed by washing three times in PBS/Triton for 5minutes each time and resuspending the cells in PBS/Triton.

Controls were prepared for each set of experiments, including control cell lines that are positive for the protein of interest (Table 2-11). A negative control for each antibody was also included, by incubating the cells in blocking buffer instead of primary antibody. This step was necessary to ensure proper quality control measure in all experiments.

2.4.3 Apoptosis assay

FITC Annexin V apoptosis detection kit I (BD Biosciences, UK) was used for identification of the different stages of apoptosis in the cells following treatment (Casciola-Rosen et al., 1996). FITC Annexin V is a sensitive probe for identifying apoptotic cells and, when used in conjunction with a vital dye such as PI (Propidium Iodide), allows the identification of early apoptotic cells (PI negative, FITC Annexin V positive). Viable cells with intact membranes exclude PI, whereas the membranes of dead and damaged cells are permeable to PI.

The cells were prepared according to manufacturer’s instructions. In brief, cells were washed twice with cold PBS and suspended in 1X Binding buffer at a concentration of 1x106 _{cells/ml. 100µl of that solution (1x10}5 _{cells) was transferred to}

an Eppendorf tube and stained with 5µl of FITC Annexin V and 5µl PI (provided in the kit), gently mixed and then incubated for 15minutes in the dark. Following this, 400µl of 1X Binding buffer was added to each sample and sample analysed by flow cytometry within 1hour.

Apoptosis percentage in cells were determined using FITC Annexin V apoptosis detection kit I (BD Biosciences, UK) (2.4.3). Several appropriate controls were added to ensure proper analysis and for quality control measures. Unstained control for each cell line was included for each experiment. Two positive controls for each cell line were also included by incubating cells in heat block for 10 seconds at 60°C, one was stained

with FITC Annexin V and one stained with PI. One untreated control was added for each cell line used to determine the baseline of normal apoptosis in cells and to ensure that the cells were not subjected to damage during experimental setting.

Samples and controls were analysed using BD AccuriTM _{C6 (BD Biosciences,}

UK) and data was analysed using BD AccuriTM _{C6 Plus software (BD Biosciences, UK).}

Gating was conducted on unstained control (Figure 2-3-A). Compensation for FITC/PI signal overlap is conducted using positive FITC control, positive PI control and mixed control (positive FITC: positive PI) (Figure 2-3-B).

Figure 2-3, Example of gating strategy used for measuring breast cancer cell lines integrity and compensation technique used to measure positive cells in each flow cytometry channel.

This figure shows gating strategy used for breast cancer cell line to exclude dead cells, debris and cellular doubling, using gated region shown in the first plot, using 10,000 events analysed each time. (A) Shows unstained breast cancer cells in forward and side scatter; also, it shows the gate of live cells. (B) Shows unstained cells in FL1 and FL2 detectors. This histogram was used to set threshold for positive cells after negative histogram. (C) The positive cells stained with propidium iodide detected in FL2. (D) The positive cells stained with primary Annexin V-FITC conjugated fluorescence antibody detected in FL1. (E) Compensation control (combination of cells stained with Annexin V-FITC conjugated fluorescence antibody only and cells stained with PI only) was used to set the compensation level for differentiating the FL1 and FL2 signal.

2.4.4 Flow cytometric technique

All flow cytometry analysis was performed using a BD AccuriTM _{C6 (BD}

Biosciences, UK) and data was analysed using the BD AccuriTM _{C6 Plus software (BD}

Biosciences, UK). A single cell population was gated based on appropriate size on the

for each sample was measured, and all experiments were repeated a minimum of three times. Increased fluorescence was compared to unstained controls for all proteins investigated. Computational analysis was performed using the BD AccuriTM _{C6 Plus}

software.

Cells were gated to exclude debris and cellular doubling, and analysis was based on collecting 10,000 events through the gate as shown in (Figure 3-3). Flow cytometry was used as well to measure the percentage of positive cells as well as the median fluorescence intensity (MFI), which provided an indication of the overall level of protein in cells. All comparisons were done by one-way analysis of variance (ANOVA) test prepared using SPSS and GraphPad Prism software for data analysis. Negative control (secondary only), positive controls (Table 2-2), and unstained cell control was included in the experiments.

Figure 2-4, Example of the gating strategy used for measuring breast cancer cell lines integrity and compensation technique used to measure the positive cells in each flow cytometry channel.

This figure shows the gating strategy used for breast cancer cell line to exclude cellular doubling, using the gated region shown in the first plot, using 10,000 events analysed each time. (A) Shows the unstained breast cancer cells in forward and side scatter, also, it shows the gate of the live cells. (B) Shows the unstained cells in FL1 detector. This histogram was used to set the threshold for the positive cells after the negative histogram. (C) The positive cells stained with the primary and secondary conjugate fluorescence antibody detected in FL1.