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

Protocolo

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.

Materiales

Name	Company	Catalog Number	Comments
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