3. Make several dilutions of the suspension and stain with BODIPY
505/515 dyes of standard concentration (0.08 μg ml
À1
).
4. Perform fluorescence reading using spectrofluorometer after
10 min of incubation in the darkness at 25
C (see Notes 6 and 7).
4 Notes
1. The phosphate buffer is used as sheath fluid only for flow cytometry analysis. Its composition is NaCl 137 mM, KCl 2.7 mM,
Na 2 HPO 4 10.0 mM, and KH 2 PO 4 1.8 mM.
2. Dimethylsulfoxide (DMSO) is among the most often used BODIPY 505/515 carriers for easier diffusion through cell membranes. The microalgae lose their vitality if culture is exposed
to 0.165 g ml
À1 (15%) of DMSO that demonstrate the toxic
effect of the solvent [16]. Organic solvents such as DMSO or
acetone should be kept to a minimum (<1%), because they
decrease BODIPY 505/515 fluorescence and give a higher
amount of cellular debris [17].
3. Although BODIPY 505/515 stains lipids in many algae species efficiently, the limited permeation of the dyes was observed in some
robust species that have thick cell wall such as Nannochloropsis
oculata [13]. Therefore, the permeation characteristic of particular
species is advised to check before the starting experiments.
4. Generally, the final concentration of BODIPY 505/515 in the
culture sample ranges between 0.04 and 2.6 μg ml
À1
. But the
optimal concentration resulting in a maximum fluorescence to
obtain optimal lipid staining for BODIPY was evaluated to be from
0.067 to 0.28 μg ml
À1 for various species, when the cell density
is around 1 Â 10
6 cell/ml. The quenching of the stain may occur
at the concentrations that are lower of optimum, whereas overstaining gives bright green fluorescence background. The optimal algal and dye concentrations should be determined for
particular microalgae species by testing several dilutions. A linear
correlation should be achieved for cell concentration and dye
fluorescence intensity [10, 17].
5. The optimal incubation temperature for BODIPY 505/515 was
found to be 25
C and the optimal incubation period—1–20 min.
Fluorescence duration should be tested for particular species for the
specification of the optimal incubation time.
6. Lipid quantification using BODIPY staining could be performed
following optimal protocol and if the relationship curves
between fluorescence and cell lipids will be established for the
particular species including different stages of its development.
7. Three different methods that can be used to evaluate the amount of
lipids in microalgae cells are listed below. (a) Fluorescence intensity of
50
Judita Koreiviene ˙
505/515 dyes of standard concentration (0.08 μg ml
À1
).
4. Perform fluorescence reading using spectrofluorometer after
10 min of incubation in the darkness at 25
C (see Notes 6 and 7).
4 Notes
1. The phosphate buffer is used as sheath fluid only for flow cytometry analysis. Its composition is NaCl 137 mM, KCl 2.7 mM,
Na 2 HPO 4 10.0 mM, and KH 2 PO 4 1.8 mM.
2. Dimethylsulfoxide (DMSO) is among the most often used BODIPY 505/515 carriers for easier diffusion through cell membranes. The microalgae lose their vitality if culture is exposed
to 0.165 g ml
À1 (15%) of DMSO that demonstrate the toxic
effect of the solvent [16]. Organic solvents such as DMSO or
acetone should be kept to a minimum (<1%), because they
decrease BODIPY 505/515 fluorescence and give a higher
amount of cellular debris [17].
3. Although BODIPY 505/515 stains lipids in many algae species efficiently, the limited permeation of the dyes was observed in some
robust species that have thick cell wall such as Nannochloropsis
oculata [13]. Therefore, the permeation characteristic of particular
species is advised to check before the starting experiments.
4. Generally, the final concentration of BODIPY 505/515 in the
culture sample ranges between 0.04 and 2.6 μg ml
À1
. But the
optimal concentration resulting in a maximum fluorescence to
obtain optimal lipid staining for BODIPY was evaluated to be from
0.067 to 0.28 μg ml
À1 for various species, when the cell density
is around 1 Â 10
6 cell/ml. The quenching of the stain may occur
at the concentrations that are lower of optimum, whereas overstaining gives bright green fluorescence background. The optimal algal and dye concentrations should be determined for
particular microalgae species by testing several dilutions. A linear
correlation should be achieved for cell concentration and dye
fluorescence intensity [10, 17].
5. The optimal incubation temperature for BODIPY 505/515 was
found to be 25
C and the optimal incubation period—1–20 min.
Fluorescence duration should be tested for particular species for the
specification of the optimal incubation time.
6. Lipid quantification using BODIPY staining could be performed
following optimal protocol and if the relationship curves
between fluorescence and cell lipids will be established for the
particular species including different stages of its development.
7. Three different methods that can be used to evaluate the amount of
lipids in microalgae cells are listed below. (a) Fluorescence intensity of
50
Judita Koreiviene ˙