5.3.1 Cultivation of Dictyostelium in liquid medium
5.3.1.1 Growth in axenic medium
Dictyostelium cells are grown under sterile condition. WT laboratory strain AX2 can grow in G0 medium, either in Costar-wells and in Petri dishes (∅ 5 cm, 10 cm) or in shaking flasks.
The incubation in the flask was under constant and gentle shaking at 150 rpm. Moreover, the culture should not exceed 1/3 of the flask volume in order to ensure an adequate oxygen supply. Culture from either dilution of a pre-culture or by thawing spores should have a minimal density of 5 x 104 cells /ml, because cultures of lower density lag for a period of time before they begin to grow. Since only cells in exponential growth phase were investigated in this work, the cells on the plates or in shaking medium were kept at a density lower than 5 x 106 cells/ml. The cell density of Dictyostelium was determined by counting with a Coulter counter. The transformants were grown in the G0 medium supplemented with appropriate antibiotic according to the introduction of the corresponding antibiotic resistance into the cells. The generation time of AX2 is about 10 h at a constant temperature of 22°C. But the generation rate may be different in the transformants.
For LD investigation, PA with 200 µM final concentration was added to axenic medium to induce TAG accumulation and LD formation. Dictyostelium cells grown in PA-containing medium are about 70% in size as compared to cells grown in normal axenic medium
(performed by H. Otto). Therefore, the quantitative assays performed in this work were normalized by total protein amount, but not the cell density.
5.3.1.2 Growth in live bacterial suspension
The suspension of two bacterial strains, Klebsiella aerogeneses (K.a) and E.coli B/r, were used for the growth of Dictyostelium WT cells. The bacterial cultures were grown overnight at 37°C in LB medium and harvested by centrifugation at 3,000 g for 15 min at 4°C. After twice wash with phosphate buffer, bacteria were resuspended in phosphate buffer at an OD600 of 8.0.
Bacteria in phosphate buffer are still alive but cannot grow, and serve as the only food source for Dictyostelium. Cells grow in bacterial suspension is faster than in axenic medium and the doubling time is approximately 4 h at 22°C and with shaking at 150 rpm. Therefore, Dictyostelium cells were inoculated into bacterial suspension at a lower density of 3 x 103 to 1 x 104/ml (final concentration), in order to avoid the depletion of food source through the overnight growth. The cell density of Dictyostelium in bacterial suspension was determined by a hemocytometer.
5.3.2 Subcloning of transformed Dictyostelium cells
Subcloning is used to clonal isolation of Dictyostelium transformation. The clones on an EP-plate were resuspended in 200-1000 µl phosphate buffer depending on the clone numbers and dilution series (1:100; 1:1000, 1:10000 - about 200 µl per set) was achieved with E.coli B/2 or K.a suspension. It is noted that E.coli B/2 cells are used for Dictyostelium growth in plastic plate while E.coli B/r are suitable for growth in shaking culture. The dilution sets were then plated out on SM plates and incubated at 22°C. After 3 days, transparent plaques were formed from the consumption of bacteria by Dictyostelium cells. The plaque is composed of spores inside formed by starvation and the vegetative cells at the edge. The vegetative cells from isolated plaques were transferred with a toothpick into 24-well Costar Plate with the appropriate selection medium and further incubated at 22°C.
5.3.3 Plaque-assay
Plaque assay is used to examine the plaque formation behaviour of Dictyostelium mutants on bacterial plates in comparison with that of WT. The density of Dictyostelium culture in shaking culture was determined by Coulter Counter. The culture was so diluted in phosphate buffer that 5 cells on average were finally inoculated in 200 µl E. coli B/2 suspension. The
mixture was then plated out on SM plate. As mentioned above (5.3.2), the plaques are visible on plate after 3 days incubation at 22°C. The diameters of the formed plaques on the SM-plates were measured with a calliper. For checking the continuity of plaque formation, the diameters of plaques were measured again at 4 days incubation. For each cell strain, plaques in three such bacterial plates were checked, so that the diameters of at least 10 plaques were measured.
5.3.4 Spore production
As mentioned before, scarce of food source induced the spore formation. Spores can be induced with this principle to preserve the cell lines. Approximate 1-1.5 x108 Dictyostelium cells from axenic medium were harvested by centrifugation at 400 g for 3 min at 4°C and washed twice with phosphate buffer. The resulting pellet was resuspended in 500 µl phosphate buffer and plated out on phosphate agar plate (∅ 10 cm), then incubated at 22°C.
The fruiting bodies were formed after two days and transferred into 1 ml phosphate buffer in cryotube with an inoculating loop. The cryotube was rapidly frozen in liquid nitrogen and stored in -70°C. To inoculate a fresh culture, the desired amount of spores were thawed (50-100 µl) in G0 medium at 22 ° C. Dictyostelium cells were visible after two day incubation and then G0 medium can be replaced by selective medium.
5.3.5 Phagocytosis assay
5.3.5.1 Preparation of TRITC-labelled yeast
Five gram of dried Saccharomyces cerevisiae were incubated in 50 ml 1 x PBS with stirring in the boiling water (in water bath) for 30 min. Then the yeasts were washed five times with 1
× PBS and twice with phosphate buffer at 1,250 g for 5 min each. The concentration of yeast was adjusted to 1 x 109 cells/ml with phosphate buffer, and the yeast could be stored at -20°C.
For the labelling with TRITC, 2 x 1010 yeasts were harvested and resuspended in 20 ml of 50 mM Na2HPO4 (pH 9.2), and added 2 mg in 200 µl of DMSO dissolved TRITC. The suspension was incubated for 30 min at 37°C with shaking. This is followed by washing twice with 50 mM Na2HPO4 (pH 9.2) and washed four times with phosphate buffer at 1,250 g for 5 min each. The concentration of the yeasts was adjusted to 1 x 109 / ml with phosphate buffer.
The TRITC-labelled yeast was transferred in 1 ml Aliquots and stored at -20°C.
5.3.5.2 Determination of phagocytic activity
This assay determine internalization rate of Dictyostelium cells by measurement of fluorescence changes over 120 min after adding the TRITC-labelled yeast cells into Dictyostelium shaking culture. To avoid adhesion of Dictyostelium cells on the glass surface, the assay was performed in previously silanized 25 ml conical flask. This was achieved by adding 15 ml silane solution in a flask under the hood and incubated for a few minutes with slight shaking. After removal of silane solution, the flask was washed with 100% ethanol and subsequently with ddH2O and then dried with lint-free tissues. After silinization treatment, the flask can be used up to three times.
This assay was performed at 22°C. 10 ml Dictyostelium cells grown in axenic medium were transferred to a silanized conical flask and kept shaking at 150 rpm. 120 µl of ultra-sonicated TRITC-labelled yeasts were added to the Dictyostelium shaking culture, and immediately 1 ml culture were transferred into a 1.5 ml reaction tube and mixed with 100 µl trypan blue ( t = 0 min). Trypan blue was used to quench the fluorescence of the yeasts in the medium (not phagocytozed). After 3 min shaking in a mixer, the sample was centrifuged at 1,000 g for 2 min. The supernatant was removed thoroughly, and the pellet was resuspended in 1 ml phosphate buffer and mixed well. The sample was measured by a fluorescence spectrophotometer (fluorimeter) with the excitation of 544 nm and the emission of 574 nm. The fluorescence was measured at a 15 min interval for total 120 min. The obtained values were normalized by protein amount (5.5.1.1) and evaluated graphically using Microsoft Excel.