13. After the fluorescence measurements, remember to consider
the “additional” dilution factor caused by DMSO (+growth
medium) additions on top of the possible initial dilution of the
culture.
14. When measuring a series of samples, add NR (and DMSO if
needed) to the samples in, e.g., 1 min intervals to samples, and
after incubation, also measure the samples using the same time
interval. Using a timer helps. For this, you need to prepare the
samples in vials and transfer the samples to the cuvette before
fluorescence measurement. This way, time is saved and all the
samples are incubated the same time.
References
1. Dutta AK, Kamada K, Ohta K (1996) Spectroscopic studies of Nile red in organic solvents
and polymers. J Photochem Photobiol A Chem
93(1):57–64
2. Murugan NA, Rinkevicius Z, A ˚ gren H (2011)
Modeling solvatochromism of Nile red in
water. Int J Quantum Chem 111:1521–1530
3. Lee SJ, Yoon B-D, Oh H-M (1998) Rapid
method for the determination of lipid from
green alga Botryococcus braunii. Biotechnol
Tech 12(7):553–556
4. Cooksey KE, Guckert JB, Williams SA, Callis
PR (1987) Fluorometric determination of the
neutral lipid content of microalgal cells using
Nile red. J Microbiol Methods 6:333–345
5. Elsey D, Jameson D, Raleigh B, Cooney MJ
(2007) Fluorescent measurement of microalgal
neutral lipids. J Microbiol Methods
68:639–642
6. Chen W, Zhang C, Song L, Sommerfeld M,
Hu Q (2009) A high throughput Nile red
method for quantitative measurement of neutral lipids in microalgae. J Microbiol Methods
77:41–47
7. Greenspan P, Fowler SD (1985) Spectrofluorometric studies of the lipid probe, Nile red. J
Lipid Res 26:781–789
8. de la Jara A, Mendoza H, Martel A, Molina C,
Nordstro ¨n L, de la Rosa V, Dı ´az R (2003) Flow
cytometric determination of lipid content in a
marine dinoflagellate Crypthecodinium cohnii.
J Appl Phycol 15:433–438
9. Satpati GG, Pal R (2015) Rapid detection of
neutral lipid in green microalgae by flow cytometry in combination with Nile red staining—
an improved technique. Ann Microbiol
65:937–949
10. Natunen K, Sepp€ al€ a J, Koivula RJ, Pellinen J
(2017) Monitoring cell-specific neutral lipid
accumulation in Phaeodactylum tricornutum
(Bacillariophyceae) with Nile red staining – a
new method for FlowCAM. J Phycol
53:396–404
11. Natunen K, Sepp€ al€ a J, Schwenk D, Rischer H,
Spilling K, Tamminen T (2015) Nile red staining of phytoplankton neutral lipids: speciesspecific fluorescence kinetics in various solvents. J Appl Phycol 27:1161–1168
12. Bligh EG, Dyer WJ (1959) A rapid method of
total lipid extraction and purification. Can J
Biochem Physiol 37:911–917
13. Chen W, Sommerfeld M, Hu Q (2011)
Microwave-assisted Nile red method for
in vivo quantification of neutral lipids in microalgae. Bioresour Technol 102:135–141
Lipid staining with Nile Red
61
the “additional” dilution factor caused by DMSO (+growth
medium) additions on top of the possible initial dilution of the
culture.
14. When measuring a series of samples, add NR (and DMSO if
needed) to the samples in, e.g., 1 min intervals to samples, and
after incubation, also measure the samples using the same time
interval. Using a timer helps. For this, you need to prepare the
samples in vials and transfer the samples to the cuvette before
fluorescence measurement. This way, time is saved and all the
samples are incubated the same time.
References
1. Dutta AK, Kamada K, Ohta K (1996) Spectroscopic studies of Nile red in organic solvents
and polymers. J Photochem Photobiol A Chem
93(1):57–64
2. Murugan NA, Rinkevicius Z, A ˚ gren H (2011)
Modeling solvatochromism of Nile red in
water. Int J Quantum Chem 111:1521–1530
3. Lee SJ, Yoon B-D, Oh H-M (1998) Rapid
method for the determination of lipid from
green alga Botryococcus braunii. Biotechnol
Tech 12(7):553–556
4. Cooksey KE, Guckert JB, Williams SA, Callis
PR (1987) Fluorometric determination of the
neutral lipid content of microalgal cells using
Nile red. J Microbiol Methods 6:333–345
5. Elsey D, Jameson D, Raleigh B, Cooney MJ
(2007) Fluorescent measurement of microalgal
neutral lipids. J Microbiol Methods
68:639–642
6. Chen W, Zhang C, Song L, Sommerfeld M,
Hu Q (2009) A high throughput Nile red
method for quantitative measurement of neutral lipids in microalgae. J Microbiol Methods
77:41–47
7. Greenspan P, Fowler SD (1985) Spectrofluorometric studies of the lipid probe, Nile red. J
Lipid Res 26:781–789
8. de la Jara A, Mendoza H, Martel A, Molina C,
Nordstro ¨n L, de la Rosa V, Dı ´az R (2003) Flow
cytometric determination of lipid content in a
marine dinoflagellate Crypthecodinium cohnii.
J Appl Phycol 15:433–438
9. Satpati GG, Pal R (2015) Rapid detection of
neutral lipid in green microalgae by flow cytometry in combination with Nile red staining—
an improved technique. Ann Microbiol
65:937–949
10. Natunen K, Sepp€ al€ a J, Koivula RJ, Pellinen J
(2017) Monitoring cell-specific neutral lipid
accumulation in Phaeodactylum tricornutum
(Bacillariophyceae) with Nile red staining – a
new method for FlowCAM. J Phycol
53:396–404
11. Natunen K, Sepp€ al€ a J, Schwenk D, Rischer H,
Spilling K, Tamminen T (2015) Nile red staining of phytoplankton neutral lipids: speciesspecific fluorescence kinetics in various solvents. J Appl Phycol 27:1161–1168
12. Bligh EG, Dyer WJ (1959) A rapid method of
total lipid extraction and purification. Can J
Biochem Physiol 37:911–917
13. Chen W, Sommerfeld M, Hu Q (2011)
Microwave-assisted Nile red method for
in vivo quantification of neutral lipids in microalgae. Bioresour Technol 102:135–141
Lipid staining with Nile Red
61