FolateRSense on the ASK


Folate receptors (FR) are a family of cell surface receptors that bind the essential vitamin folic acid (folate, vitamin B9). These receptors are upregulated in highly metabolic cells due to the high need of this vital nutrient. FolateRSense™ 680 is a near infrared fluorescent agent that can be used to noninvasively quantify FR expression in vivo. It binds specifically to the folate receptor in vivo and can be used to image folate receptor expression.

Figure 1: FolateRSense 680 mechanism of action. FolateRSense 680 is a fluorescent targeted agent that specifically binds to the folate receptor alpha (FRA) protein- a biomarker for tumor metabolism.

Products and catalog numbers

ProductCatalog NumberEx/Em wavelength (nm)Molecular weight (g/mol)Validated ExperimentsApplicationsStorage and Stability
FolateRSense 680NEV10040670/6901605.7In vivo/Ex vivo
Flow cytometry
In vitro microscopy
Acute Inflammation
Technical Data Sheet

Using FolateRSense 680 in vivo/ex vivo

In Vivo imaging

The generally recommended procedure for in vivo imaging with FolateRSense 680 is administration via intravenous injection and imaging 6-24 hours post injection.View instructions on setting up an in vivo mouse experiment with FolateRSense 680 and imaging on an IVIS or FMT system.

Route of InjectionMouse Dose (25 g)Rat Dose (250 g)Blood t 1/2Tissue t 1/2Optimal imaging timeOptimal Re-injection Time (complete clearance)Route of Metabolism/ background tissueFMT and IVIS settings
IV2 nmol6-20 nmol5 min>168 h6 h (6-24 h)6-7 daysKidneysFMT 680/700
IVIS 675/720
Figure 2: Biodistribution and Pharmacokinetics A) KB cells were injected into the mammary fat pads of Nu/Nu female mice. Mice were injected intravenously with 2 nmoles FolateRSense 680 once tumors reached the desired size. Tissue samples were collected 4 hours later and fluorescence assessed by planar imaging (ex vivo). Mean counts/energy for each tissue were determined as a measure of tissue brightness. B) The pharmacokinetic profile was assessed by injecting CD1 mice, then collecting plasma at multiple times post-injection and measuring the plasma fluorescence. The t ½ was determined to be 5 minutes.

Figure 3: In Vivo Imaging and Quantification A) KB cells were injected into the mammary fat pads of Nu/Nu female mice. Once tumors reached the desired size, the mice were injected intravenously with 2 nmoles FolateRSense 680 and imaged by tomographically (top) and by reflectance (bottom) at 2 hours and then at different times thereafter. Panels show two representative mice. B) Quantification of the tumor site signal (pmoles by tomography) and relative tumor brightness (mean counts/energy by reflectance) were determined over time.

Figure 4: In Vivo Imaging and Quantification. Mice were implanted with KB, HeLa or human colorectal HT-29 tumors. A) Once tumors reached the desired size, mice were injected with FolateRSense 680 in the absence or presence of 100x excess folic acid and imaged tomographically 4 hours later. Four representative mice per group are shown. B) The quantification of the signal in the different tumors with and without competitive folic acid is shown.

Ex Vivo Imaging:

Figure 5: Ex Vivo Imaging. KB, HeLa or HT-29 tumors-bearing mice were injected with FolateRSense 680, with or without excess folic acid as a competitor. The tumors were excised 4 hours later and then snap frozen. Using fluorescence microscopy, labeling of the tumor cells and effective competition by free folic acid can be seen. The FITC-conjugated anti-folate receptor antibody validates the results in the lower panel. In blue: DAPI nuclear stain, red: FolateRSense 680, green: FITC-anti-folate receptor antibody. The acquisition time was the same for FolateRSense 680 with and without the free folic acid.

FolateRSense 680 in vitro

Flow cytometry and in vitro microscopy

We have validated FolateRSense 680 for use with fluorescence microscopes and flow cytometers. Here is a brief protocol with a recommended concentration of agent to use:

  1. Culture tumor cells (such as KB) in standard TC plate or chamber slide 
  2. Incubate cells with 1 uM FolateRSense 680 for 1 hour at 37 °C 
  3. Wash 3 times in PBS 
  4. Flow cytometry filter settings: 712/21
    Fluorescence micrsocopy filter: Cy 5.5

In Vitro Imaging:

Figure 6: Fluorescence Microscopy and Flow Cytometry. KB or HeLa cells were incubated with 1mM FolateRSense 680 in the presence or absence of excess unlabeled Folic acid (FA) for 1 hour at 37 °C. After washing, cells were analyzed by fluorescence microscopy and flow cytometry. A) The fluorescence microscopy shows labeling of positive cells and the second panel shows FolateRSense 680 is effectively competed off by free folic acid. In the third panel, a FITC-conjugated anti-folate receptor antibody was used to validate the results. In blue: DAPI nuclear stain, red: FolateRSense 680, green: FITC-anti-folate receptor antibody. B) The flow cytometry results confirm significantly higher labeling of folate receptor positive cells over negative cells. The binding experiments using increasing concentrations of FolateRSense 680 were used to determine an approximate dissociation constant of 5 nM.

Application notes and posters

  • Poster: Quantitative assessment of folate receptor expression by FMT imaging with a NIR fluorescent folate agent


Q. Can I use FolateRSense 680 in humans?

A. No, FolateRSense 680 is intended for animal research only and not for use in humans.

Q. What cancer cell lines have Folate receptors overexpressed?

A. In a variety of cancers (including breast, ovarian, lung, kidney, colon, and brain) and in tumor associated macrophages, folate receptors (FR) are overexpressed. Folic acid has been successfully used as a cancer specific targeting moiety for the efficient delivery of chemotherapeutic agents and diagnostic reporters. The determination of the level of FR expression for a given tumor is critical to the success of such agents, and therefore, specific and quantitative imaging agents and methods for the determination of FR expression in vivo are imperative.

Q. Does FolateRSense 680 bind to Folate Receptor alpha or beta?

A. The R&D team has tested the specificity of this agent for Folate receptor protein. We have not done studies to show whether it specifically binds to alpha or beta or both.


Please visit our Citations Library for references using FolateRSense 680 on the IVIS or on the FMT.