Cell viability assay - Cell Titer Blue ® (CTB assay)

where D represents the applied radiation dose and α and β are proportionality factors. The dose-modifying factor (DMF) was calculated by the ratio of the radiation dose in the absence or presence of CXCL12 and AMD3100 to achieve the same cell survival rate.

2.3.6 Cell viability assay - Cell Titer Blue

(CTB assay)

TheCTB assay is a cell viability assay that provides a homogeneous, fluorometric method to monitor cell viability and cell metabolic activity in multiwell plates. The assay is based on a redox reaction. Viable, metabolically active cells, can convert resazurin (dark blue with little intrinsic fluorescence activity) into its highly fluorescent product, resorufin (pink, highly fluorescent molecule) (Promega, Technical Bulletin, revised 6/09, Figure 2.1). The intensity of the fluorescence is proportional to the number of viable cells and their metabolic activity.

Sterile 96-well black plates with clear bottoms were used for analysis.

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Figure 2.1: Conversion of resazurin with little fluorescence activity to resorufin with highly fluorescence activity by viable cells. The intensity of the fluorescence produced is proportional to the number of viable cells and their metabolic activities. (The figure was taken from Promega product-guide-page: www.promega.de/resources/product -guides-and-selectors/protocols-and-applications-guide/cell-viability)

2.3.6.1 Determination of optimal incubation time and radiation dose within CTB assay

Test 1: Optimal incubation time

Before setting up the 96-well plates for the cell viability assay, four different incubation times (26, 48, 72, and 96 hours) were tested to find the best possible incubation time. This experiment was performed only for control cell line HaCat and tumour cell line ZMK-1.

First, a suspension of HaCat and ZMK-1 cells was prepared and maintained in a 15 ml plastic tube. One tube contained a suspension of the control cells without irradiation; after preparing the cell suspension, the second tube was irradiated. The irradiation was performed as described in Chapter 2.3.5.2. A total dose of 4 Gy was delivered with a dose rate of 1 Gy/min.

After the irradiation the cells were seeded into wells; 5000 cells per well for HaCat and 6000 cells per well for ZMK-1 (see Table 2.12, green area).

48 Test 2: Optimal radiation dose

Suspensions containing the necessary number of cells were prepared and maintained in 15 ml plastic tubes. One tube contained cells for the control group (no irradiation); the other tubes were irradiated. After delivering with a dose rate of 2 Gy/min total doses of 2, 4, 6, 8, 10 and 12 Gy the cells were seeded into wells, with 5000 cells/well for HaCat and 6000 cells/well for ZMK-1 (see Table 2.12, green area).

Test 3: Combination of optimal incubation time and radiation dose

Due to non-optimal incubation times for observing the effect of irradiation on cell viability, the assay was repeated for all five cell lines with a longer incubation time, i.e. for one week.

For this test, a radiation dose of 8 Gy was selected as the optimal dose. For observing the effect of irradiation on cell viability, we decided to reduce the cell density to 2000 cells per well. In this last test, the cells were seeded in wells and the plate was then irradiated. Two plates were prepared; one 8 Gy irradiated plate, and one non-irradiated plate.

With the purpose of completing and homogenizing the volume per well, a total volume of 100 μl of each cell suspension per well was added to each well. The outer wells on all sides were loaded with cell-free medium (cell-free medium: M, Table 2.12). The contents of the wells in the first column (column 1, blue area B1-G1) were used as blank samples to determine background fluorescence. The green area represents the different conditions investigated. For each experiment and condition the test was done in triplicate (Table 2.12).

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

Table 2.12: Schematic r epresentation of a 96-well plate seeded with cells and medium

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The prepared plates (irradiated and non-irradiated) were incubated for one week under standard incubation conditions (37°C and 5% CO2). At the end of the incubation period, the plate was removed from the incubator, 20 μl of CTB Reagent was added cells in culture as well as 6 wells in the column 1 (Blank wells) following the manufacturer’s guides, and the plate incubated again for one hour at 37°C before reading the 96-well plate on a fluorometer at 560 nm excitation and 590 nm emission.

2.3.6.2 Investigating various treatments on cell viability

The notion of optimal radiation dose and incubation time permits to optimise the experimental process. If more treatment combinations are required in the same experiment, e.g. irradiation, CXCL12 and/or AMD3100, one plate is not irradiated and the other is irradiated allowing each treatment condition to be analysed with and without irradiation.

To begin with, a cell suspension containing 2 x 10⁴ cells/ml was prepared and divided in four 15 ml plastic tubes. The first tube was treated with 100 ng/ml of CXCL12, a second tube with 5 µg/ml of AMD3100, a third tube with both of them. The last tube did not contain any drug.

All tubes were incubated for 30 minutes at 37°C and 5% CO2. From each tube 100 µl of the cell suspension (2000 cells/well) was pipetted in two sterile 96-well black plates with clear bottoms; each experimental condition was performed in triplicate. One of this 96-well-plate was irradiated. A total dose of 8 Gy was delivered with a dose rate of 2 Gy/min.

After a one-week incubation time, the plates were removed from the incubator and CellTiter-Blue^® Reagent was added to the wells. The plates were gently shaken for ten seconds and returned to the incubator for one more hour at 37 °C for one hour. The experiment was performed twice for each cell line and the data were analysed following the procedure described below (see following Chapter 2.3.6.3).

2.3.6.3 Data analysis

The fluorescence analysis was performed with a Wallec1420 VICTOR^TM plate reader. The dye was excited with a 560 nm wavelength and the emissions measured at 590 nm. The obtained data were imported into Microsoft Office and the following calculations were performed: background fluorescence was subtracted from the raw fluorescence results of all

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wells. The averages and the standard deviations of the triplicate determinations were calculated.