Nile Red Staining: A Technique to Detect and Quantify Intracellular Neutral Lipids in Algae

Published: April 30, 2023

Abstract

Source: Storms, Z. J. et al. A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence. J. Vis. Exp. (2014)

This video demonstrates a protocol for detecting and quantifying neutral lipids in algal cells by staining them with Nile red dye.

Protocol

1. Fluorometric Quantification of Neutral Lipids Using Nile Red

NOTE: Only 10 µl of an algal suspension at 5 g/L is needed for the fluorescence reading. Generally, isolation of dry algal biomass from 1.5 ml of culture broth is more than sufficient. Also, the light intensity of the lamp in the spectrophotometer can degrade over time. It is recommended to include standards in every experiment to ensure that variations in the instrument do not add unnecessary error to the measurements.

  1. Prepare a Nile Red solution at a concentration of 10 µg/ml dissolved in alcohol reagent grade ethanol. Store this solution in the dark at 4 °C.
  2. Prepare a 30% (v/v) ethanol solution in deionized water and store at 4 °C.
  3. Prepare all algal samples at the same biomass concentration (5 g/L is recommended) and in the same manner as the standards used in the measurement. Do this by either suspending pre-dried samples in the appropriate amount of phosphate buffer (0.6 g/L potassium phosphate dibasic, 1.4 g/L potassium phosphate monobasic), or adjusting the concentration of a growing algal culture to 5 g/L with phosphate buffer after measuring the turbidity.
    NOTE: Measurements performed on live algal cultures will often have larger error associated with them depending on the precision of the turbidity calibration curve. Resuspending dried samples may require the use of a homogenizer to fully disperse the biomass.
  4. For each sample, mix 80 µl of the 30% ethanol solution, 10 µl of the Nile Red solution, and 10 µl of algal suspension in a single well of a 96-well plate. In order to properly account for the variability of the fluorescence measurement, perform 5 replicates of each sample.
  5. Run a two-point calibration curve with standards in order to account for day-to-day variations in the instrument and preparation. Prepare the standards for fluorescence measurement using the same procedure as the samples.
    NOTE: Generally, two points is sufficient for recalibration of the instrument, three points can be run to verify linearity.
  6. Perform the fluorescence measurements in a multi-well plate reader spectrophotometer. The following conditions were found to yield the most consistent results:
    1. Shake at 1,200 rpm, orbit 3 mm, for 30 sec.
    2. Incubate at 40 °C for 10 min.
    3. Shake at 1,200 rpm, orbit 3 mm, for 30 sec.
    4. Record fluorescence, excitation at 530 nm, emission at 604 nm.
  7. Convert the fluorescence measurements to oil content using the results from the internal standards.

Divulgazioni

The authors have nothing to disclose.

Materials

25 ml disposable pipettes Fisher  13-676-10K
Pipette Bulb  Fisher  13-681-51
1.5 ml microcentrifuge tubes  Fisher  05-408-129
40 ml Centrifugation tubes (FEP)  Fisher  05-562-16A  Could also use glass tubes
Pasteur glass pipettes  Fisher  13-678-20C
Nile Red  Sigma  N3013-100MG
Ethanol (alcohol reagent grade)  Fisher  AC65109-0020
Leica DMRXA2 (or equivalent) microscope Leica  DMRXA2
Fluorescence multi-well plate reader  Thermo Lab Systems  Fluoroskan Ascen
Fluorescence reader software  Thermo Lab Systems  Ascent Software 2.6
COSTAR 96 well plate with round bottom  Fisher  06-443-2

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Citazione di questo articolo
Nile Red Staining: A Technique to Detect and Quantify Intracellular Neutral Lipids in Algae. J. Vis. Exp. (Pending Publication), e21032, doi: (2023).

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