MTT assay. The activity of the active compound in inhibit fungal and bacterial biofilm was quantified by determining biofilm cell viability using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. MTT (Sigma) solution (5 mg/mL in PBS) was prepared, filter-sterilized using a 0.22- µm pore size filter. The preformed biofilms were first washed three times with 200 µL PBS, and then 100 µL of PBS buffer and 20 µL of the MTT solution were added to each of the prewashed xxxxx. The microtiter plate was then incubated for 2 h at 37⁰C. Following incubation, the PBS-MTT solutions were removed from the xxxxx and replaced with 100 µL of DMSO in each well to dissolve the water soluble purple formazan crystal. The color intensity of the soluble formazan was determined using microplate reader (Bio-Rad 680 XR) at 550 nm. Percentage of cell survival is expressed as the formula below: 𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑐𝑒𝑙𝑙 𝑠𝑢𝑟𝑣𝑖𝑣𝑒 = 100% x absorbance of treated cells absorbance of control cells Qualitative analysis on C. albicans biofilm A Carl Zeiss LSM 5 Exciter Laser Scanning Confocal Microscope (Leica Microsystems, Germany) was used to observe the architecture of the C. albicans biofilms in the presence of massoia lactone. Candida biofilms were formed by dispensing standardized cell suspension (500 µL of a suspension containing 1.0 x 107 cells/mL in LB medium) onto 13 mm diameter pre-sterilized plastic coverslips (Thermanox; Nulge Nunc International) placed in the xxxxx of pre-sterilized flat bottomed twelve- well plates (Iwaki). The plate was incubated at 37ºC for 90 minutes (adhesion phase). Following incubation period, the supernatant was removed and a total of 500 µL RPMI medium containing different concentrations of massoia lactone (100 – 6.25 µg/mL) were added to the washed xxxxx. The plate then incubated for 8 h (adherence phase), 24 h (intermediate phase) and 48 h (maturation phase) at 37 ºC. The coverslips then were washed twice with PBS and stained using the LIVE/DEAD fluorescent stains (10 µL of 3.34 µM SYTO9 and 10 µL of 20 µM Propidium Iodide (PI) both in DMSO) (Molecular Probes, USA) before examined under CLSM. Serial sections in the xy plane were obtained at 1 μm intervals along the z axis. A 20× and 40x oil immersion objective were used with 488 nm Ar laser excitation and 500–640 nm band pass emission setting. The image were subsequently analysed using the freely available image processing software imageJ version 1.46 (Xxxxxxx, National Inst...
MTT assay. Following incubation as described in Section 2.4.2, the ability of the cells to reduce MTT to formazan was assessed as a measure of cell viability (Xxxxxxx et al. 2009). Ten µL of MTT solution (table 2.3) was added to each well, with the exception of the 2 no MTT control xxxxx, using a repeat pipettor. The plate was then placed in the incubator for 4 hours. Media was carefully aspirated from all the xxxxx and 100 µL of lysis buffer (table 2.3) was added. The plate was then placed inside an Orbital Incubator SI50 (Stuart Scientific, Stone, UK) at 150 rpm for 1 hour at 37oC, after which absorbance was measured at 570nm using a spectrophotometer (Xxxxxx Xxxxx, Massachusetts, USA). Results were calculated and expressed as described in section 2.5.4.
MTT assay. To evaluate cell proliferation of MSC and measure the number of viable cells, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium) reduction assay was performed. MTT is a tetrazolium compound which penetrates viable cells (Xxxx, Xxxxxxx et al. 2016) . First, cells were seeded in 96-well plates and treated with growth media. For assays 25 mg of MTT powder was dissolved in 25 ml of PBS at 37°C. Using normal medium without Ascorbic Acid, a %10 dilution of MTT solution was made and filtered. 100µl per well of this solution was added to the cells and incubated at 37°C for 3 hours. When crystals were visible under the microscope, the solution was removed from xxxxx by gentle tapping onto a wad of tissues and replaced by 100µl per well of DMSO. Plates were then left on a shaker at medium speed for 5 minutes. The plate was transferred to a microplate reader machine to measure its absorbance using Quicklink program with reference wavelength of 630nm and absorbance maximum 570 nm. MTT data were analysed by two-way ANOVA with Xxxxxxxxxx’s post hoc test to determine whether time and treatment had any statistically significant effect on cells viability. Statistical analysis was performed using Prism 7.04 software (Graph Pad Software Inc.). Data are expressed as means ± S.D and the statistical significance is set at p<0.05.
MTT assay. Tumour cells were incubated with T-cells at specified effector to target (E:T) ratios. In the case of adherent targets, residual tumour cell viability was quantified using an MTT assay at 48 and/or 72 hours. After removal of the supernatant and residual T-cells, MTT (Sigma, Poole, UK) was added at 500 μg/mL in D10 medium for 40 minutes at 37°C and 5% CO2. Formazan crystals were resuspended in DMSO and absorbance was measured at 560 nm.
