Technical Note 179

Fluorescein Performance Data

Introduction

Fluorescein and its derivative fluorescein isothiocyanate (FITC) are ubiquitous in cell and molecular biology as a fluorescent label for cells and in many other applications. Fluorescein is a fluorescent compound that is easily detected using a fluorometer or fluorescence microscope. Fluorescein has an excitation maximum at 490 nm and an emission maximum at 514 nm.

DeNovix QFX Fluorometer and DS-11 Series Spectrophotometer / Fluorometers enable precise fluorescein quantification using a proprietary optical fluorescence core and four user-selectable LED channels. .The data presented in this note was measured in the Basic Fluorometer app of EasyApps software.

Materials and Methods

A fluorescein dilution series diluted from a fluorescein standard (ThermoFisher Scientific cat #F36915) was quantified on a DeNovix QFX fluorometer.

The 50 µM standard was diluted in 0.05 M borate buffer with pH 8.5. A dilution series with 12 fluorescein concentrations between 0 nM and 2500 nM was created by serial dilution. Sample volumes of 200 µL were measured in 0.5 mL PCR tubes (DeNovix cat #TUBE-PCR-0.5-500). Measurements were made in the Basic Fluorometer app using the blue channel. The blue channel excitation range is 442 – 497 nm, and the emission range is 514 – 561 nm.

Results

The fluorescein standard curve from 0 to 500 nM is shown in Figure 1. Linearity was exhibited between 0.05 and 2500 nM fluorescein. Table 1 shows the raw data for the full dilution series.

Table 1: Fluorescein Raw Data through Full Concentration Range
Fluorescein Concentration (nM)RFU (n = 3)StDev%CV
0116.670.800.7
0.05123.290.990.8
0.1131.210.380.3
0.25155.950.620.4
0.5196.840.330.2
1.0274.671.230.4
5901.251.900.2
101699.353.650.2
254087.3915.740.4
10023394.9533.140.1
500113476.45190.030.2
1000221854.98507.980.2
2500534489.981459.920.3
Figure 1: Fluorescein linearity through the concentration range of 0 – 500 nM.

Revised 19 Oct 2020