3.2 Basic Nile Red
Staining Protocol for
Monitoring Lipid
Accumulation
1. Take a fresh sample from your culture and dilute the sample if
necessary (see Note 5).
2. Prepare a series of samples, where you have a 3 mL blank
sample (i.e., growth medium, see Note 5), and parallel 3 mL
(diluted) culture samples. If you are using DMSO (e.g., 10%
(v/v)), you have a 2.7 mL blank sample and 2.7 mL culture
samples.
3. Add chosen volume (see Sect. 3.1) of NR stock solution one by
one into all the samples (i.e., blank + culture samples) (see Note
14). If you are using DMSO (e.g., 10% (v/v)), add 0.3 mL
DMSO and chosen volume of NR one by one into all the
samples (i.e., blank + culture samples).
4. Let the stained samples incubate in darkness (see Note 1) for
the chosen time.
5. Measure NR fluorescence of each sample at the chosen wavelengths (see Note 10).
6. To obtain a proxy for cell-specific lipid concentration in the
culture, divide the obtained fluorescence values by the culture’s
current cell concentration (cells/mL).
7. Carry out the measurements daily to follow lipid accumulation
and choose the best days for harvesting the lipids. Figure 2
shows the results of monitoring cell-specific lipid accumulation
in duplicate Phaeodactylum tricornutum cultures with NR fluorescence measurements and GC.
4 Notes
1. NR is photosensitive, i.e., the fluorescence intensity is quenched
when exposed to bright light. Therefore, also samples stained
with NR should be incubated in darkness.
2. Do not use technical grade NR.
3. When using acetone as a solvent, always close the stock solution
bottle well, and minimize the usage and storage time to prevent
solvent evaporation. When working with NR dissolved in
DMSO, a more stable solvent, wear special safety gloves, as
DMSO easily penetrates through the skin.
4. It is important that the cuvette is suitable for fluorescence
measurements. Do not use plastic cuvettes.
5. For checking the linear range of the instrument, prepare a
dilution series and measure and determine the linear range of
the fluorescence output. If the sample is outside the linear
range, a dilution step must be added. If the growth medium
Lipid staining with Nile Red
59
Staining Protocol for
Monitoring Lipid
Accumulation
1. Take a fresh sample from your culture and dilute the sample if
necessary (see Note 5).
2. Prepare a series of samples, where you have a 3 mL blank
sample (i.e., growth medium, see Note 5), and parallel 3 mL
(diluted) culture samples. If you are using DMSO (e.g., 10%
(v/v)), you have a 2.7 mL blank sample and 2.7 mL culture
samples.
3. Add chosen volume (see Sect. 3.1) of NR stock solution one by
one into all the samples (i.e., blank + culture samples) (see Note
14). If you are using DMSO (e.g., 10% (v/v)), add 0.3 mL
DMSO and chosen volume of NR one by one into all the
samples (i.e., blank + culture samples).
4. Let the stained samples incubate in darkness (see Note 1) for
the chosen time.
5. Measure NR fluorescence of each sample at the chosen wavelengths (see Note 10).
6. To obtain a proxy for cell-specific lipid concentration in the
culture, divide the obtained fluorescence values by the culture’s
current cell concentration (cells/mL).
7. Carry out the measurements daily to follow lipid accumulation
and choose the best days for harvesting the lipids. Figure 2
shows the results of monitoring cell-specific lipid accumulation
in duplicate Phaeodactylum tricornutum cultures with NR fluorescence measurements and GC.
4 Notes
1. NR is photosensitive, i.e., the fluorescence intensity is quenched
when exposed to bright light. Therefore, also samples stained
with NR should be incubated in darkness.
2. Do not use technical grade NR.
3. When using acetone as a solvent, always close the stock solution
bottle well, and minimize the usage and storage time to prevent
solvent evaporation. When working with NR dissolved in
DMSO, a more stable solvent, wear special safety gloves, as
DMSO easily penetrates through the skin.
4. It is important that the cuvette is suitable for fluorescence
measurements. Do not use plastic cuvettes.
5. For checking the linear range of the instrument, prepare a
dilution series and measure and determine the linear range of
the fluorescence output. If the sample is outside the linear
range, a dilution step must be added. If the growth medium
Lipid staining with Nile Red
59