microscopy [7], and flow cytometry [4, 8, 9]. Recent evidence
shows that neutral lipids can also be quantified at a cell-specific
level by measuring the NR fluorescence of stained cells with the
image-based particle analyzer FlowCAM™ [10]. This opens new
possibilities to study the effect of species diversity on lipid accumulation, as with the FlowCAM species with distinct morphologies
can be distinguished from each other, and species-specific properties can be determined.
This method description focuses on spectrofluorometric NR
fluorescence measurement of microalgae neutral lipids. The basic
staining principle is relatively simple, but obtaining the different
staining parameters requires optimization, as most of them are both
species- and system-specific [11]. Therefore, I start by describing
an optimization protocol to help choose the optimal staining parameters for each new species (see Sect. 3.1) and, based on that, a
basic staining protocol to be applied for daily monitoring of lipid
accumulation (see Sect. 3.2). An alternative to the NR method, lipid
staining with another fluorescent dye, BODIPY
® 505/515, is presented in another chapter in this book.
2 Materials
Prepare and store all reagents at room temperature. The NR stock
solution needs to be stored in darkness (see Note 1). Follow your
local waste disposal regulations when disposing waste materials.
1. Microalgae culture where neutral lipid production has been
induced by, e.g., nitrogen limitation.
2. Solvents: acetone and dimethyl sulfoxide (DMSO) (see Sect.
3.1).
3. Ethanol (96%).
4. Nile red (NR) stock solution: Prepare a 0.25 mg/mL NR stock
solution by dissolving NR powder (see Note 2) in acetone or in
DMSO (see Note 3).
5. Spectrofluorometer and quartz cuvette(s) (10 mm light path,
volume 3 mL) suitable for fluorescence measurements
(see Note 4).
3 Methods
Carry out all procedures at room temperature. After using NR,
rinse cuvette and all other reusable glassware with ethanol to
remove traces of the dye. When measuring fluorescence, the fluorescence values should always stay in the linear range of your
spectrofluorometer (see Note 5).
56
Katariina Natunen
shows that neutral lipids can also be quantified at a cell-specific
level by measuring the NR fluorescence of stained cells with the
image-based particle analyzer FlowCAM™ [10]. This opens new
possibilities to study the effect of species diversity on lipid accumulation, as with the FlowCAM species with distinct morphologies
can be distinguished from each other, and species-specific properties can be determined.
This method description focuses on spectrofluorometric NR
fluorescence measurement of microalgae neutral lipids. The basic
staining principle is relatively simple, but obtaining the different
staining parameters requires optimization, as most of them are both
species- and system-specific [11]. Therefore, I start by describing
an optimization protocol to help choose the optimal staining parameters for each new species (see Sect. 3.1) and, based on that, a
basic staining protocol to be applied for daily monitoring of lipid
accumulation (see Sect. 3.2). An alternative to the NR method, lipid
staining with another fluorescent dye, BODIPY
® 505/515, is presented in another chapter in this book.
2 Materials
Prepare and store all reagents at room temperature. The NR stock
solution needs to be stored in darkness (see Note 1). Follow your
local waste disposal regulations when disposing waste materials.
1. Microalgae culture where neutral lipid production has been
induced by, e.g., nitrogen limitation.
2. Solvents: acetone and dimethyl sulfoxide (DMSO) (see Sect.
3.1).
3. Ethanol (96%).
4. Nile red (NR) stock solution: Prepare a 0.25 mg/mL NR stock
solution by dissolving NR powder (see Note 2) in acetone or in
DMSO (see Note 3).
5. Spectrofluorometer and quartz cuvette(s) (10 mm light path,
volume 3 mL) suitable for fluorescence measurements
(see Note 4).
3 Methods
Carry out all procedures at room temperature. After using NR,
rinse cuvette and all other reusable glassware with ethanol to
remove traces of the dye. When measuring fluorescence, the fluorescence values should always stay in the linear range of your
spectrofluorometer (see Note 5).
56
Katariina Natunen