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Nile Red assay development for the estimation of neutral lipids in Chlorella emersonii and Pseudokirchneriella subcapitata

Priyanka Priyanka
Priyanka Priyanka
, 
Gemma K. Kinsella
Gemma K. Kinsella
, 
Gary T. Henehan
Gary T. Henehan
 and 
Barry Ryan
Barry Ryan
   | Oct 21, 2020
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Article Category: Research Article
Published Online: Oct 21, 2020
Page range: 216 - 222
DOI: https://doi.org/10.2478/ebtj-2020-0025
© 2020 Priyanka Priyanka, Gemma K. Kinsella, Gary T. Henehan, Barry Ryan, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Figure 1

The effect of different concentrations (10-60% v/v) of DMSO on the fluorescence intensity of neutral lipids from Chlorella emersonii. A constant concentration of Nile Red was maintained (10μg/ml) for all the solvents. Different excitation wavelengths resulted in different fluorescence intensities for both types of solvent with 530nm being the wavelength for maximum fluorescence intensity from both solvents after 30mins of incubation at room temperature. Data represented here are the average of three independent experiments, with five assay values each, and the error bars shown are the standard deviation.
The effect of different concentrations (10-60% v/v) of DMSO on the fluorescence intensity of neutral lipids from Chlorella emersonii. A constant concentration of Nile Red was maintained (10μg/ml) for all the solvents. Different excitation wavelengths resulted in different fluorescence intensities for both types of solvent with 530nm being the wavelength for maximum fluorescence intensity from both solvents after 30mins of incubation at room temperature. Data represented here are the average of three independent experiments, with five assay values each, and the error bars shown are the standard deviation.

Figure 2

The effect of different concentrations (10-60% v/v) of acetone on the fluorescence intensity of neutral lipids from Chlorella emersonii. A constant concentration of Nile Red was maintained (10μg/ml) for all the solvents. Different excitation wavelengths resulted in different fluorescence intensities for both types of solvent with 530nm being the wavelength for maximum fluorescence intensity from both solvents after 30mins of incubation at room temperature. Data represented here are the average of three different experiments, with five assay values each, and the error bars shown are the standard deviation.
The effect of different concentrations (10-60% v/v) of acetone on the fluorescence intensity of neutral lipids from Chlorella emersonii. A constant concentration of Nile Red was maintained (10μg/ml) for all the solvents. Different excitation wavelengths resulted in different fluorescence intensities for both types of solvent with 530nm being the wavelength for maximum fluorescence intensity from both solvents after 30mins of incubation at room temperature. Data represented here are the average of three different experiments, with five assay values each, and the error bars shown are the standard deviation.

Figure 3

The effect of different concentrations (10-60% v/v) of DMSO on the fluorescence intensity of neutral lipids from Pseudokirchneriella subcapitata . A constant concentration of Nile Red was maintained (10μg/ml) for all the solvents. Different excitation wavelengths resulted in different fluorescence intensities for both types with 530nm being the wavelength for maximum fluorescence intensity from both solvents after 30mins of incubation at room temperature. Data represented here are the average of three independent experiments, with five assay values each, and the error bars shown are the standard deviation.
The effect of different concentrations (10-60% v/v) of DMSO on the fluorescence intensity of neutral lipids from Pseudokirchneriella subcapitata . A constant concentration of Nile Red was maintained (10μg/ml) for all the solvents. Different excitation wavelengths resulted in different fluorescence intensities for both types with 530nm being the wavelength for maximum fluorescence intensity from both solvents after 30mins of incubation at room temperature. Data represented here are the average of three independent experiments, with five assay values each, and the error bars shown are the standard deviation.

Figure 4

The effect of different concentrations (10-60% v/v) of Acetone on the fluorescence intensity of neutral lipids from Pseudokirchneriella subcapitata. A constant concentration of Nile Red was maintained (10μg/ml) for all the solvents. Different excitation wavelengths resulted in different fluorescence intensities for both types with 530nm being the wavelength for maximum fluorescence intensity from both solvents after 30mins of incubation at room temperature. Data represented here are the average of three independent experiments, with five assay values, each and the error bars shown are the standard deviation.
The effect of different concentrations (10-60% v/v) of Acetone on the fluorescence intensity of neutral lipids from Pseudokirchneriella subcapitata. A constant concentration of Nile Red was maintained (10μg/ml) for all the solvents. Different excitation wavelengths resulted in different fluorescence intensities for both types with 530nm being the wavelength for maximum fluorescence intensity from both solvents after 30mins of incubation at room temperature. Data represented here are the average of three independent experiments, with five assay values, each and the error bars shown are the standard deviation.

Figure 5

Fluorescence intensity from neutral lipids of Chlorella emersonii and Pseudokirchneriella subcapitata utilizing different dye concentrations (1-50μg/ml) in 20% (v/v) of DMSO, with an excitation wavelength of 530nm and an emission wavelength of 580nm. Microalgal strains were incubated at 40°C for 60mins before measuring fluorescence intensity. The data represented here are the average of three independent experiments, with five assay values each, and the error bars shown are the standard deviation.
Fluorescence intensity from neutral lipids of Chlorella emersonii and Pseudokirchneriella subcapitata utilizing different dye concentrations (1-50μg/ml) in 20% (v/v) of DMSO, with an excitation wavelength of 530nm and an emission wavelength of 580nm. Microalgal strains were incubated at 40°C for 60mins before measuring fluorescence intensity. The data represented here are the average of three independent experiments, with five assay values each, and the error bars shown are the standard deviation.

Figure 6

The effect of incubation time on the fluorescence intensity of neutral lipids from Chlorella emersonii and Pseudokirchneriella subcapitata when microalgal cells were incubated at 40°C with 10μg/ml and 5μg/ml of Nile Red dye in 20% (v/v) of DMSO respectively. A maximum fluorescence intensity in Chlorella emersonii was observed after 60mins of incubation and for Pseudokirchneriella subcapitata 40mins of incubation was ideal. Data represented here are the average of three independent experiments, with five assay values each, and the error bars shown are the standard deviation.
The effect of incubation time on the fluorescence intensity of neutral lipids from Chlorella emersonii and Pseudokirchneriella subcapitata when microalgal cells were incubated at 40°C with 10μg/ml and 5μg/ml of Nile Red dye in 20% (v/v) of DMSO respectively. A maximum fluorescence intensity in Chlorella emersonii was observed after 60mins of incubation and for Pseudokirchneriella subcapitata 40mins of incubation was ideal. Data represented here are the average of three independent experiments, with five assay values each, and the error bars shown are the standard deviation.

Figure 7

The relationship between fluorescence intensity and number of cells (OD590nm) when Chlorella emersonii and Pseudokirchneriella subcapitata were stained with 10μg/ml and 5μg/ml Nile red in 20% (v/v) DMSO respectively. Fluorescence was recorded after 40min and 60min of static incubation at 40°C respectively. Data represented here are the mean of three independent experiments and the error bars shown are the standard deviation.
The relationship between fluorescence intensity and number of cells (OD590nm) when Chlorella emersonii and Pseudokirchneriella subcapitata were stained with 10μg/ml and 5μg/ml Nile red in 20% (v/v) DMSO respectively. Fluorescence was recorded after 40min and 60min of static incubation at 40°C respectively. Data represented here are the mean of three independent experiments and the error bars shown are the standard deviation.

Figure 8

Microscopic observation of Chlorella emersonii under (a) 40X and (b) 100X magnification. Microalgal cells containing neutral lipids present in Chlorella emersonii after incubation with 20% (v/v) DMSO with 10μg/ml of Nile Red dye under 100X magnification (c). Cells without any fluorescence have either no neutral lipid or too few lipids to be observed visually.
Microscopic observation of Chlorella emersonii under (a) 40X and (b) 100X magnification. Microalgal cells containing neutral lipids present in Chlorella emersonii after incubation with 20% (v/v) DMSO with 10μg/ml of Nile Red dye under 100X magnification (c). Cells without any fluorescence have either no neutral lipid or too few lipids to be observed visually.

Figure 9

Microscopic observation of Pseudokirchneriella subcapitata under (a) 40X and (b) 100X. Microalgal cells containing neutral lipids present in Pseudokirchneriella subcapitata after incubation with 20% (v/v) DMSO with 5μg/ml of Nile Red dye under 100X magnification (c). Cells without any fluorescence have either no neutral lipid or too few lipids to be observed visually.
Microscopic observation of Pseudokirchneriella subcapitata under (a) 40X and (b) 100X. Microalgal cells containing neutral lipids present in Pseudokirchneriella subcapitata after incubation with 20% (v/v) DMSO with 5μg/ml of Nile Red dye under 100X magnification (c). Cells without any fluorescence have either no neutral lipid or too few lipids to be observed visually.

Figure 10

Triolein standard curve, used for the quantification of neutral lipids in Chlorella emersonii and Pseudokirchneriella subcapitata.
Triolein standard curve, used for the quantification of neutral lipids in Chlorella emersonii and Pseudokirchneriella subcapitata.

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