2 Chapter Two Materials and Methods
2.14 Random Mutation of D.armatus Cells
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2.13.3 Flow Cytometry Analysis
The analysis of D. armatus cells staining with Nile Red were carried out using a high-speed flow cytometer BD LSRII (BD Bioscience, Figure 2.8) based on (Velmurugan et al., 2013). To obtain the fluorescence readings, the Nile Red stained cells at different concentrations were excited with a 475-nm laser, and the emission was 568/42 nm while the unstained cells were utilised as an auto-fluorescence control. The features of the D. armatus cells including cell size and granularity using flow cytometry were obtained by Forward scatter (FSC) and side scatter (SSC) signals. Flow cytometry data were analysed depending on many parameters including the stained cells grandparents’
percentage, and median of the stained cells using Flow Jo software.
Figure 2.8 High speed flow cytometry BD LSRII Flow cytometry
2.14 Random Mutation of D.armatus Cells
Induced random mutations using Ultra Violet (UV) light were used to attempt to increase the lipid content of D. armatus cells based on method described by (Bougaran et al., 2012). Well grown D. armatus cells (OD595 approximately 1) were irradiated for different time periods (3, 6, 9, 12, 16, 32, 48, 60 min) using a UV – C lamp at 254 nm
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(Upland, CA 91786 USA). The UV exposure was achieved by adding 12 ml of the D.
armatus culture into a 90-mm sterile Petri dish (Thermo Scientific), the plate was transferred under the UV lamp and after taking off the lid of the Petri dish the distance between the lamp and the dish was about 13.5 cm.
Three replicates were prepared for each irradiation time. The cells were transferred into pre-autoclaved 100 ml BG11 medium flasks. To minimise the probability of photoreactivation that would lead to photorepair of UV-Induced damage, the flasks were kept in dark for 24 h. The OD595 and cell count for each flask were measured directly after exposure and over the next 8 days. The samples were then incubated for 30 days. Then, the neutral lipid content of the mutated cells was analysed as described in 2-13-3 using cells at OD 1.5 =106, and Nile Red concentration at 128 µg/ml.
2.14.1 UV Mutant Cells Sorting
After the data of UV mutant cells were analysed, the optimum condition cells were sorted using (BD FACS AriaTM IIU, Figure 2.9) based on (Terashima et al., 2015). The optimum condition of samples were re-exposed to UV at the time that achieved the highest mean of stained cells as described in section 2-13-3. Then the samples were re-prepared as described in section 2-14-1 but with one concentration of Nile Red (the concentration that gave the highest mean of stained cells). After that, 1 ml of the unstained sample (control) was run through a high-speed flow cytometry to make a gate. The spreading of stained cells was exploited as a control to fix the high lipid gate for expected mutant cells. The upper 3% of the stained cells was fixed as a high lipid gate. Then, all cells that chop down the gate were accumulated into sterile vials containing 1 ml autoclaved BG11 medium.
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Figure 2.9 BD FACS Aria for sorting of mutant cells
2.14.2 Selection of Mutated Cells
The sorted cells were transferred to 96 well plate according to (Lim et al., 2015) for the selection of single mutated cells. Single cell cloning serial dilution technique was used to achieve single sorted cells. All the wells of the plate filled with 100 µl of BG11 medium.
1000 sorted cells were transferred from the sorted tube to the first well of the plate (A1) to the final volume of 200 µl. then 100 µl was transferred from well A1 to B1 after mixing well, the steps were continued to the well H1, then 100 µl of the mixture was discarded from H1 well. 100 µl of the diluted cells were transferred to the A2 wells (A2 to H2) and then transferred consequently to all wells of the plate. Finally, 100 µl of the diluted cells were transferred to (A1-H1) wells of the plate from A12-H12) wells. The same single cell technique was conducted with the wild type cells to be exploited as a control. Then the plates were incubated at 25 ± 1OC with continuous light (50 – 70 µmol m-2 s-1). All the cells in each well was tested on the next day using an inverted microscope (Olympus CK40 culture microscope) to differentiate the wells that contain single cells and label the well. After the single cells were grown well, the growth was transferred to a 12-well plate containing 2 ml BG11 medium. After the cells grown the well. After the single cells were grown well, the growth was transferred to a 12-well they were
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transferred into a 250-ml flask containing 100 ml BG11 medium. The cells were grew for a month, and subsequently the neutral lipid content of 13 single UV mutated cells and the wild type cells (used as control) were analysed using Flow cytometry after staining with 1 µg/ml and followed the same steps of flow cytometry analysis of neutral lipid described in section 2.13.2 and 2.13.3.
2.14.3 Nile Red determination of the wild type and UV exposed D.armatus. neutral lipid
The neutral lipid of both wild type and UV exposed D. armatus cells grown on both normal BG11 and N free BG11 for 14 days were determined using Nile red dye as described in section 2-9-2-2-1 using an optimum concentration of cells (OD595=50%=200 µg ml-1 Dry weight) of cells and Nile Red concentration (1 µg ml-1), the neutral lipid concentration and percentage of lipid were determined as described in section 2.10.
2.15 statistical analysis
Comparison of mean values of highest stress condition against the normal contions were conducted using Excel T test, in all cases, comparisons that showed a p value <0.05 were deemed significant.
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