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Fluorescence Measurements and Instruments

Fluorescence instruments, such as fluorometers enable researchers to quantify analytes and their reactions with a high degree of accuracy and selectivity. Unlike UV/VIS spectrophotometers, which detect anything absorbing at 260 nm, including contaminants, fluorometers employ target-specific dyes to quantify biomolecules, such as nucleic acids, proteins, or endotoxins, in microliter (µL) sample volumes. The high-binding affinity of these dyes for their intended target, followed by a significant increase in their fluorescence upon binding, allows for a more precise measurement of the target despite contamination or low target concentration. Because of its sensitivity and ease of use, fluorescence dye-based quantification is routinely used for initial sample quantification in numerous molecular applications, including cloning, sequencing, transfection, qPCR, lipopolysaccharide (LPS) detection, and gel electrophoresis.

The CytoCite™ BG100 Portable Fluorometer is a user-friendly, single-channel instrument designed to work seamlessly with the Portelite™ assay kits. Together they provide a comprehensive solution for fast, sensitive, and accurate quantification of specific nucleic acid species, proteins, and endotoxins using a simple mix-and-read format.

 

 

CytoCite™ Benchtop Fluorometers



CytoCite™ Portable Fluorometer
The CytoCite™ BG100 Portable Fluorometer is a cloud-integrated, benchtop device that accurately measures ssDNA, dsDNA, total nucleic acids, RNA, proteins, and endotoxins using the highly sensitive Portelite™ quantitation kits. With a small footprint, the CytoCite™ BG100 and CytoCite™ GR100 devices are the most compact fluorometers available on the market, facilitating easy integration into any laboratory, point-of-care use, or field-testing environment. As the only commercially available cloud-enabled fluorometer, the CytoCite™ Fluorometer offers several significant advantages relative to other instruments, such as the Qubit® Fluorometer (ThermoFisher). Features include unlimited data storage, automated daily data backups to prevent unexpected data loss, and access to the Quest Graph™ analytics suite featuring a set of fully integrated tools for fast and easy regression modeling, IC50 calculation, and more.

The CytoCite™ BG100 Fluorometer utilizes sample volumes of as little as 1 µL and are several orders of magnitude more sensitive than absorbance-based instruments, such as the Nanodrop™ (ThermoFisher) for both DNA (absorbance = 260 nm) and protein (absorbance = 280 nm) quantification. The CytoCite™ BG100 instrument is optimized to detect probes in the ubiquitously employed FITC channel (Ex/Em = 480 nm/520 nm), with an overall limit of detection of as little as 1 nM of fluorescein. For red fluorescence-emitting probes (Ex/Em = 530 nm/620 nm), such as resorufin-based assays, the CytoCite™ GR100 instrument is recommended.
 

Table 1. The following table lists the physical, electrical, and optical properties of the CytoCite™ Fluorometer

Instrument
CytoCite™ Fluorometer
Dimensions3.54"L x 2.83"W x 1.54"H (9.0 cm x 7.2 cm x 3.9 cm)
Weight~0.3 lbs (135 g)
Operating Ranges 10 - 30 °C; < 80% relative humidity
Installation location Indoor use only
Typical Power Consumption 2.5 VA
Power Requirements 5 VDC, 0.5 A
Computer Interface Micro-USB B, USB 2.0
Dynamic Range 5 orders of magnitude
Light source (device dependent) Blue LED (max ~470 nm) (Cat No. CBG100)
Green LED (max ~530 nm) (Cat No. CGR100)
Excitation Filter (device dependent) Blue 457 - 487 nm (Cat No. CBG100)
Green 517 - 547 nm (Cat No. CGR100)

 

Portelite™ Quantitation Assays Optimized for the CytoCite™ and Qubit® Fluorometers


Unlike conventional UV absorbance, which can overestimate sample concentrations due to contaminants in the sample such as salts, solvents, detergents, proteins, free nucleotides, the Portelite™ assays utilize target-selective dyes that emit fluorescence when bound to DNA (ssDNA, dsDNA), RNA, endotoxins, and proteins.

Portelite™ Nucleic Acid Quantitation Kits



DNA standard curve generated using Portelite™ Fluorimetric DNA High Sensitivity Quantitation Kit (Cat No. 17661). Fluorescence intensity was quantified using the FITC channel, and the regression model was calculated using log-log best-fit. The detection limit was 10pg/µL.
Portelite™ Nucleic Acid Quantitation Kits, optimized for the CytoCite™ BG100 Fluorometer, are designed to selectively quantify total nucleic acid, DNA (ssDNA, dsDNA), and RNA, providing more sensitive analysis than comparable UV absorbance-based instrumentation. All assays are performed using the same general protocol, providing a simple mix-and-read assay format for minimal hands-on time. Each Portelite™ kit contains a concentrated assay reagent, dilution buffer, and pre-diluted standards. The fluorescent probes in each kit have similar spectral properties, being blue light-excitable (480 nm), with emissions between 510 nm and 580 nm. While developed specifically for use with the CytoCite™ BG100 fluorometer, Portelite™ Fluorometric kits may also be used on other commercially available compact fluorometric instruments, such as the Qubit® fluorometer. In addition to the Portelite™ Quantitation Kits described above, we offer Portelite™ Kits for quantifying endotoxins and proteins.

Advantages of Portelite™ Fluorimetric Quantitation Kits

  • Enhanced selectivity - assays utilize target-specific dyes that bind selectively to ssDNA, dsDNA, total nucleic acid, or RNA
  • High sensitivity - requires as little as 1 µL of sample for quantitation
  • Wide dynamic range - fluorescence-based assays with 4 to 5 orders of magnitude linear dynamic range (10pg/µL to 1 µg/µL DNA)

 

Table 2. Portelite™ Fluorimetric Nucleic Acid Quantitation Kits for use with the CytoCite™ and Qubit® fluorometers.

Product
Platform
Ex/Em (nm)
Target
Detection Range
Unit Size
Cat No.
Portelite™ Fluorimetric High Sensitivity DNA Quantitation KitCytoCite™ BG100 or Qubit® Fluorometer480/520dsDNA0.2-100 ng100 tests17660
Portelite™ Fluorimetric High Sensitivity DNA Quantitation KitCytoCite™ BG100 or Qubit® Fluorometer480/520dsDNA0.2-100 ng500 tests17661
Portelite™ Fluorimetric DNA Quantitation Kit with Broad Dynamic RangeCytoCite™ BG100 or Qubit® Fluorometer480/530dsDNA2-1000 ng100 tests17665
Portelite™ Fluorimetric ssDNA Quantitation KitCytoCite™ BG100 or Qubit® Fluorometer480/530ssDNA1-200 ng200 tests17625
Portelite™ Fluorimetric Total Nucleic Acid Quantitation KitCytoCite™ BG100 or Qubit® Fluorometer480/530dsDNA, ssDNA, RNA, oligosas little as 0.3 ng200 tests17631
Portelite™ Fluorimetric RNA Quantitation KitCytoCite™ BG100 or Qubit® Fluorometer480/530RNA5-100 ng100 tests17658
Portelite™ Fluorimetric RNA Quantitation KitCytoCite™ BG100 or Qubit® Fluorometer480/530RNA5-100 ng500 tests17659


Portelite™ Fluorimetric Protein Quantitation



Serial dilutions of BSA, chicken-egg ovalbumin, porcine thyroglobulin were measured at Ex/Em = 485/590 nm using the Portelite™ Fluorimetric Protein Quantitation Kit *Optimized for CytoCite™ and Qubit® Fluorometers* with Qubit® Fluorometer. As low as 50 ng/mL of protein can be detected.
Quantification of protein concentration represents one of the most widely used methods in biomedical research, being particularly applicable to research involving protein extraction, purification, labeling, crystallization, and analysis. Accurate protein quantification is essential to a range of other critical assays that require precise results for total protein content to generate reliable data. For example, when comparing different protein extracts by SDS-polyacrylamide gel electrophoresis, accurate protein quantitation is necessary to ensure equal amounts of samples are present in each lane. The Portelite™ Fluorimetric Protein Quantitation Kit (11109, 11111) is a rapid and precise fluorescence-based analytical procedure that measures protein concentration in a solution. The assay provides a dynamic range of 12.5 mg/mL to 5 mg/mL when using bovine serum albumin (BSA) as the calibration standard. The kit is more convenient and consumes less precious samples than conventional colorimetric solution assays for protein quantification, such as the bicinchoninic acid (BCA), Bradford (Coomassie Blue), and Lowry assays. The kit is suitable for estimating protein concentration in measuring protein purity from column fractions after chromatography or samples to be analyzed by electrophoresis and a host of other common research applications. The kit is optimized for performance on the Cytocite™ and Qubit® fluorometers.
 

Portelite™ Fluorimetric Endotoxin Quantitation



E.coli endotoxin dose-response was measured in a 0.2 mL, thin-wall PCR tube using the CytoCite™ BG100 in the green fluorescent channel. As low as 0.002 EU/mL of E.coli endotoxin can be detected with 10 minutes incubation.
Lipopolysaccharides (LPS), also called endotoxins, are large molecules consisting of a lipid and polysaccharide composed of O-antigen, outer core, and inner core joined by a covalent bond. Endotoxins are found in the outer membrane of Gram-negative bacteria, and they can bind the CD14/TLR4/MD2 receptor complex in many cell types, particularly in monocytes, dendritic cells, macrophages, and B cells, which subsequently promotes secretion of various pro-inflammatory mediators, leading to septic shock. Detection of contaminating LPS in biopharmaceuticals, particularly protein, peptide, and antibody preparations, is a crucial activity in the context of biomanufacturing and bioprocessing. Portelite™ Rapid Fluorimetric Endotoxin Detection Kit (60008, 60009) uses Endotoxin Green™, a sensitive fluorogenic substrate to detect endotoxin. Endotoxin Green™ is hydrolyzed in the presence of endotoxins and the Limulus Amebocyte Lysate (LAL), an extract of blood cells obtained from horseshoe crabs, to produce a strong green fluorescence signal. The endotoxin activity is proportional to the fluorescence intensity resulting from the hydrolysis of Endotoxin Green™. The kit is optimized for use on CytoCite™ and Qubit® fluorometers and can detect a broad range of endotoxin (from 1 EU/ml to 0.002 EU/ml) present in biological samples.
 

Product Ordering Information


 

Table 3. Ordering information for the CytoCite™ fluorometers and Portelite™ assays for nucleic acid, protein, and endotoxin quantitation in solution.

Product
Unit Size
Cat No.
CytoCite™ BG100 Portable FluorometerFluorometerCBG100
CytoCite™ GR100 Portable FluorometerFluorometerCGR100
CytoCite™ Sample Tube500 TubesCCT100
Portelite™ Fluorimetric DNA Quantitation Kit with Broad Dynamic Range *Optimized for Cytocite™ and Qubit® Fluorometers*100 Tests17665
Portelite™ Fluorimetric High Sensitivity DNA Quantitation Kit *Optimized for CytoCite™ and Qubit® Fluorometers*100 Tests17660
Portelite™ Fluorimetric High Sensitivity DNA Quantitation Kit *Optimized for CytoCite™ and Qubit® Fluorometers*500 Tests17661
Portelite™ Fluorimetric Protein Quantitation Kit *Optimized for CytoCite™ and Qubit® Fluorometers*100 Tests11109
Portelite™ Fluorimetric Protein Quantitation Kit *Optimized for CytoCite™ and Qubit® Fluorometers*500 Tests11111
Portelite™ Fluorimetric RNA Quantitation Kit*Optimized for Cytocite™ and Qubit® Fluorometers*100 Tests17658
Portelite™ Fluorimetric RNA Quantitation Kit*Optimized for Cytocite™ and Qubit® Fluorometers*500 Tests17659