FAQs

The dose range lies between 2-3.5% DSS. If you have not run any studies before, we recommend testing a few animals at 2%, 2.5%, and 3% DSS.

Although DSS is highly stable both in its solid state and in aqueous solutions, its stability can be influenced by various factors. For instance, maintaining an elevated pH (e.g., over 8) or heating solutions above 70-80℃ for extended periods can lead to the dissociation of sulfate groups in the solution. When it comes to photostability, using foil to protect long-standing stock solutions is advisable. However, during experiments, such protective measures are not mandatory, as noticeable changes in DSS properties typically require prolonged exposure to ambient light.

Dissolving DSS in hard water can cause cloudy solutions or precipitations. Use soft water or ultra-pure water if the problem appears.

The bottle containing the dextran powder should be tightly closed. Although dextran is very stable, exposure to humidity can cause it to turn into a gel, making it difficult to handle. Therefore, it is important to keep the sample well-sealed. For fluorescent products, the containers should preferably be stored in a dark place, away from direct ambient light.

For solutions prepared in PBS buffer, they can be stored in the refrigerator at 4°C for a couple of weeks. However, it is recommended to aliquot the solution and keep the stock in the freezer at -20°C or lower for longer-term storage, lasting several months. When needed, the sample can be thawed, diluted to the required concentration, and stored in the refrigerator at 4°C for up to a couple of weeks.

The best solvents for dextran are water, followed by DMSO and formamide. For higher molecular weight dextran samples, it is necessary to heat the mixtures to up to 70°C with vigorous stirring to obtain clear solutions. Once clear solutions are formed, they can be cooled down to room temperature without precipitation.

We offer both high-sulfated (HS) and low-sulfated (LS) dextran sulfate products in a wide range of molecular weights. Both types exhibit anticoagulant properties that are affected with the degree of sulfation: greater sulfation enhances anticoagulation. However, high sulfation also increases (cyto)toxicity. So, the selection between high or low sulfated dextran sulfates depends on the application, cell line, or other experimental design parameters.

Regarding molecular weight (MW), higher MW can lead to increased toxicity. The trend of aggregation and anticoagulant capacity remains consistent at higher MWs. The choice of MW is typically influenced by other experimental parameters; for example, a solution of higher MW dextran sulfate at a certain concentration will have significantly higher viscosity than a solution of a lower MW product. Viscosity often affects the experimental setup, leading to the use of smaller amounts of high MW product in the medium to achieve a solution of lower viscosity. However, this would reduce the anticoagulant capacity of the medium, which is influenced by the concentration of the anticoagulant.

Yes, TdB labs can manufacture alkyne modified dextrans as well as azide-dextrans upon customers’ request according to the desired molecular weight of dextran, degree of substitution and the exact type of modification required.

In lysine-dextran (LD), the typical degree of substitution is approximately one lysine for every ten glucose units. This implies that for a 10kDa LD product, there would be approximately six lysine units attached.

Yes, blue dextran of high molecular weight significantly increases the viscosity of the solution.

Blue dextran is highly dipolar, containing both primary and secondary amino groups, anionic sulfonates (at a pH of about 6-7), and hydroxy groups from the dextran backbone. Consequently, blue dextran is likely to interact with small dipolar molecules capable of efficient hydrogen bonding, such as amino acids, alcohols, amines, and carboxylic acids.

The settling time of blue dextran is medium-dependent, meaning that highly viscous or thick solutions will slow down the settling process.

Controlling the exact number of charged entities per glucose unit is challenging. Our protocol, which yields approximately one charged group every four glucose units, is well-optimized and results in low batch-to-batch variability. While it is possible to produce Q-substituted dextran derivatives with different substitution levels, achieving precise control to every 3, 5, 7, or 8 units is not guaranteed. Each new protocol requires validation to ensure the same low degree of variability and high accuracy.

There may be sodium chloride present in Q-dextran, as it is used during production. However, we cannot determine its concentration since we do not analyze it.

Charge density is dictated by the degree of substitution of a batch of Q-Dextran. For instance, if there are 5 mmol of functional groups per kg of product QD1000 Da, then there will be 5 mmol of charged species per mole of product (since 1000 Da equals 1 mole). Essentially, this implies that there are as many positive charges as there are functionalities.

We exclusively produce Q-dextran, that has the highest charge density amongst our products.

TRITC-dextran is delivered at pH between 6.5 and 7.0 at which it appears to be neutral. The powder should always be kept at room temperature, but once it is resuspended, it should be stored at 4°C for a short term up to 7 days and at -20°C for a long term up to a few weeks. It is however very important to always store samples in the dark.

No. TRITC-dextran has an excitation maximum at 550 nm and an emission maximum at 571 nm at pH 9. So, the interference of TD with FITC is not very pronounced, yet there might be some. If it is strictly irradiated at 550 nm, FITC should not be excited as it exhibits a relatively narrow fluorescence and excitation band, the latter centered at 495 nm i.e. 60 nm away. However, if there is an urge to avoid any interference, its recommended to use a deep-red emitting dye like ATTO647N^TM instead of TRITC which can be excited at ca. 645 nm and fluoresce pure-red light at around 665 nm.

To obtain a calibration line for TRITC-dextran, it is preferable to use the conditions which resemble the most to the experimental setup since various components of the medium like proteins and enzymes could influence the photophysics of the fluorescent dye by photoreacting with it.

The charge of the macromolecule strongly depends on the pH. In TLD500, there are fewer TRITC fluorophore units than lysine units, so the charge is primarily determined by lysine. Given that the isoelectric point of lysine is approximately 9.7, and assuming this holds for lysine dextran at physiological pH (7.4), lysine dextran is acidic, meaning its N-termini are protonated, resulting in a net positive charge. To achieve a net neutral charge, the experiment should be conducted under buffered conditions close to pH 9.7. If such a high pH is not favorable, using TRITC-dextran instead of TRITC-lysine-dextran is recommended.

Recently prepared solutions of TD4 can be safely stored between 2 and 8ºC for one-two weeks in the dark.

The degree of substitution (DoS) of all TRITC products is higher than 0.001 which means you have at least one fluorophore every 1000Da of molecular weight. When these derivatives are produced and analyzed, the DoS pertaining to that specific batch is mentioned in the certificate of analysis you receive.

While comparing different TRITC-dextrans, if the degree of substitution (DoS) of one sample is 0.002, it implies two times more fluorophores per mass of product than in the case of 0.001. In that case, to achieve the same results in terms of emission intensities, you would need to use a higher concentration of dextran with lower DoS.

Spectrophotometry is the recommended technique used for determining the degree of substitution of both lysine and FITC in lysine-dextran and FITC-labeled products, including FITC-lysine-dextran respectively. The amount of primary amino group within lysine-dextran is determined using a Fluram method where fluorescamine is examined which is proportional to the amino content. FITC substitution within FITC-labeled products is quantified based on the absorbance measurement and subsequent calculation using an extinction coefficient derived from a standard plot.

The difference between DS10HS and DS10LS is the sulfur content. HS stands for High Sulfation (16-20% S w/w) whereas LS corresponds to Low Sulfation (8-13% S w/w). FITC-dextran sulfate is dextran sulfate sodium labeled with FITC, where the sulfur content is between 16 to 19%. So, the control for FITC-dextran sulfate 10kDa would be DS10HS. However, FDS10LS could also be produced as a CSP in case of request, and then DS10LS could be used as a non-FITC control.

DSS40 could be used as a control for FITC-dextran sulfate 40 kDa.

Two groups of charged FITC-dextran derivatives can be used for mass transfer studies:

– Polyanionic fluorescent labelled dextrans: FITC-CM-dextran and FITC-dextran sulfate

– Polycationic fluorescent labelled dextrans: FITC-DEAE-dextran and FITC-Q-dextran [Q-dextran is “purely” cationic derivative meaning that all functionalized moieties are positively charged irrelevant of the pH, while DEAE-dextran is partly cationic meaning that you can expect uncharged basic (amine) moieties as well as positively charged (tetralkyl-ammonium) moieties bound to dextran].

FITC-dextran (especially MW=2MDa) is difficult to dissolve in acetic acid and chloroform. DMSO (dimethylsulfoxide) is an organic solvent that solubilizes dextran derivatives and would solubilize FITC-dextran 2MDa as well. However, its high boiling point could be a problem. So, an alternative solution is formamide (HCONH2) or to use mixtures of water and DMSO or water and HCONH2. However, formamide also has a high boiling point, but it is possible to remove. To facilitate the dissolution of large macromolecules, heating can be applied but ultrasonication should be avoided since it could result in the FITC dissociation.

Yes, it is possible. After collecting the mouse tissue and homogenizing it, the sample should be stirred in warm water (e.g. 60-75℃) to leach out FITC-dextran in the water solution. Repeating this process two-three times (until stable fluorescence is achieved) will extract nearly all quantities of the fluorescent product. The aqueous solutions should be combined, and the fluorescence should be measured with a fluorometer. The fluorescence can then be compared to a standard solution of FITC-dextran of known concentration. It is important to note that FITC will remain bound to dextran during this procedure, so the leached-out FITC will not be free. To ensure all bound and unbound FITC is extracted, it is better to use ethanol instead of water. Additionally, some fluorophores might get damaged during the extraction process by using this or the other method, so there is no guarantee that all FITC units will remain chemically unchanged during the experiments.

It is recommended to sterilize FITC-CM-dextran in its powder form rather than as a stock solution, as heating or sterilizing the solution may cause the fluorophore units to detach. However, if you need to sterilize the solution, filter sterilization is also effective.

All our FITC derivatives can be excited at λ=495 nm, which can be conveniently achieved using a 488 nm laser line, resulting in very bright green fluorescence.

FITC-dextran 70 is highly soluble in water and can be dissolved in pure water at a temperature of 50℃ and at a concentration of up to 20g/100ml. The concentration prepared, however, depends on the purpose of the experiments. Typically, very high concentrations are prepared for stock solutions, which should be stored in the dark (e.g., by covering the vials with aluminum foil) and used as fresh as possible. These stock solutions should not be stored for more than 1-2 weeks.

Depending on the application, the minimum quantity of FITC that can be used in 100ml of solution varies from a few milligrams to some grams.

It is recommended to prepare a 20-25mM stock solution of FITC-dextran 70 (i.e., 10g in 100ml). Then, dilute 10µl of this stock solution with 990µl of the buffer of your choice. This will reduce the concentration of FITC-dextran by a factor of 100, resulting in an approximate concentration of 200µM, which can be passed through the filters without clogging them.

The degree of fluorescent labeling is close to 0.005 mole of fluorescein per mole of fructose unit. Accordingly, there are estimated 125 mmol FITC labels per mole of inulin that has a molecular weight of 4kDa.

A variety of buffers can be used for dissolving FITC-inulin, but the most appropriate ones are the borate buffer and phosphate buffer.

Generally, inulin and its derivatives are not readily soluble in water at room temperature. To facilitate the dissolution of FITC-inulin in aqueous buffer, heating the solution up to 70℃ is recommended.

The concentration of FITC-inulin is the most significant factor influencing its aggregation. Highly concentrated solutions tend to aggregate more. Introducing a small amount of DMSO can often help to dissolve these aggregates. However, the use of DMSO must be compatible with the experimental design and requirements.

We perform sequential purification steps of FITC-inulin to ensure the removal of unbound FITC dye. The free-FITC dye content at each step is checked using chromatography, ensuring that every released batch contains less than 100 ppm of the dye, as specified in our CoA. One of our product’s strengths in the market is our ability to quantify the free-dye content, which is typically in the range of 10 ppm or lower, well below the CoA requirement. This minimizes the probability of non-specific effects due to the presence of free dye.

Recently prepared solutions of FD4  can be safely stored between 2 and 8ºC for one-two weeks in the dark.

No. TRITC-dextran has an excitation maximum at 550 nm and an emission maximum at 571 nm at pH 9. So, the interference of TD with FITC is not very pronounced, yet there might be some. If it is strictly irradiated at 550 nm, FITC should not be excited as it exhibits a relatively narrow fluorescence and excitation band, the latter centered at 495 nm i.e. 60 nm away. However, if there is an urge to avoid any interference, its recommended to use a deep-red emitting dye like ATTO647N^TM instead of TRITC which can be excited at ca. 645 nm and fluoresce pure-red light at around 665 nm.

FITC-dextran is typically injected intravenously when studying blood-brain barrier (BBB) permeability. Some of these studies include:

1. Blood-brain barrier integrity in a mouse model of Alzheimer’s disease with or without acute 3D6 immunotherapy: This study explores the administration of 4 kDa FITC-dextran to assess BBB permeability in an Alzheimer’s disease model.

2. High and low molecular weight fluorescein isothiocyanate (FITC)–dextrans to assess blood-brain barrier disruption: Technical considerations: A technical examination of using FITC-dextran for assessing BBB disruption.

3. Radiation-induced permeability and leukocyte adhesion in the rat blood–brain barrier: Modulation with anti-ICAM-1 antibodies: This article investigates radiation-induced changes in BBB permeability, employing FITC-dextran as a tracer.

4. Modulation of blood-brain barrier permeability by neutrophils: In vitro and in vivo studies: Research exploring how neutrophils affect BBB permeability, using 4 kDa FITC-dextran.

5. Differential effects of hydrocortisone and TNFα on tight junction proteins in an in vitro model of the human blood-brain barrier: This paper assesses how tight junction proteins affect BBB permeability using FITC-dextran. For accurate results, intravenous administration is generally recommended for BBB studies.

However, there are studies that have used oral administration to assess BBB permeability. Some of these studies include:

1. Changes in brain kynurenine levels via gut microbiota and gut-barrier disruption induced by chronic ethanol exposure in mice: This study shows FITC-dextran 4 kDa in plasma after oral administration, highlighting gut-barrier disruption.

2. Oral Administration of Lactobacillus Inhibits the Permeability of Blood‐Brain and Gut Barriers in a Parkinsonism Model: This study assesses the effects of oral administration of FITC-dextran 4 kDa on BBB permeability in a Parkinsonism model.

3. Assessment of gut barrier integrity in mice using fluorescein-isothiocyanate-labeled dextran: This paper describes FITC-dextran 4 kDa administered orally to examine permeability in gut barrier models, which may extend to BBB studies.

All our stability studies are performed at pH levels above 4, and FITC-dextran is quite stable in the pH range of 4-9. While there is no stability data available for FITC-dextran at pH levels lower than 4, at elevated pH levels (above 9), there is a possibility of the dye dissociating from dextran. Published articles suggest that FITC-dextran 4 is stable in the gut environment of mice, as it could be tracked after 8 hours in the gut, indicating it could pass the acidic environment intact. FITC-dextran 4 is soluble in water and aqueous media. It is also soluble in acidic medium, but in simulated gastric acid with very low pH (1.5-3), the dye might dissociate from dextran depending on the pH and time, resulting in a much lower signal. Laboratory experiments are needed to verify its stability in acidic media after dissolving.

The difference between these products lies in the raw material, dextran. We use dextran with different molecular weights, corresponding to different degrees of polymerization. As the degree of polymerization (the number of glucose units in a polymer) increases, the molecular weight also increases. Therefore, in FITC-dextran 70, the dextran used has a higher molecular weight than in FITC-dextran 4. To put it simply, 4 kDa means the average molecular weight of the dextran molecules is 4,000 Daltons, while 70 kDa means the average molecular weight is 70,000 Daltons. The increase in molecular weight is thus due to the longer polysaccharide chains in the higher molecular weight dextrans.

Yes, cationic dextrans can be labelled by our Antonia Red^TM dye.

Sometimes, the components of the medium in which Antonia Red-dextran (ARD) is dissolved can interact with the fluorophore, affecting the fluorescence intensity. Additionally, the concentration of ARD can be an important factor, as increasing its concentration in a solution does not necessarily lead to an increased fluorescence.

Also, for all fluorophores, quenching can occur through various pathways. Increasing the degree of dye substitution can potentially raise fluorescence intensity, but achieving higher values can be challenging. An alternative to the red-fluorescent Antonia Red is to use another red-emitting dye, such as ATTO647N.

The recommended dosage is 1 mg of ARLD (or any dextran) in 100 µL of either PBS or saline per mouse. ARLD is highly stable under these conditions. Higher concentrations are not advised unless the fluorescence intensity is lower than expected, in which case the concentration can be tentatively increased to 2 mg of ARLD in 100 µL of PBS/saline.

Yes, this can be achieved upon customers’ request.

FITC-dextran can be used for this purpose and depending on the customers’ requirement, its molecular weight can be customized. Also, if the DNA is already tagged with FITC, to prevent confusion in imaging, another kind of fluorescent dye such as ATTO390^TM-dextran (blue emitting dye with max. absorbance at 390 nm and fluorescence centered at 476 nm) and ATTO425^TM (blue emitting dye with max. absorbance at 439 nm and fluorescence centered at 485 nm) can be used.

The selection of an appropriate molecular weight of labeled dextran for studies on endocytosis is highly dependent on the specific type of endocytosis being investigated. There are several points to be considered:

-For probing general fluid-phase endocytosis, including macropinocytic and micropinocytic processes, small dextrans could be used e.g. Dextran 10kDa.

-For merely macropinocytic processes, Dextran 70kDa is better.

Moreover, the type of fluorescent labeling is also an important parameter to be considered. For example:

Green fluorescence:

-FITC-dextran 10kDa or 70kDa (excitation/emission: 495nm/519nm)

-ATTO488^TM-dextran 10kDa or 70kDa (excitation/emission: 500nm/520nm)

Red fluorescence:

-TRITC-dextran 10kDa or 70kDa (excitation/emission: 550nm/575nm)

-Antonia Red^TM-dextran 10kDa or 70kDa (excitation/emission: 583nm/602nm)

“Deep” red fluorescence:

-ATTO647N^TM-dextran 10kDa or 70kDa (excitation/emission: 646nm/664nm)

The fluorescent labeling for all our products is always covalent. In FITC, AR, and TRITC cases, this is achieved through the formation of a thiocarbamate linker.

Yes, we have developed pH probes, dextrans labeled with dual fluorophores for e.g. FITC-TRITC-dextran and FITC-Antonia Red-dextran to achieve accurate pH determination in cells and tissue samples.

Texas Red is a “classic” solution for applications requiring the absence of carboxylates, amides, amines etc. Its attachment to dextran sulfate is possible as a customized dextran derivative upon the customers’ request.

For fixation studies, using a dextran functionalized with amino groups, such as lysine-dextran, is recommended. Lysine has two amino groups: one vicinal to a carbonyl (more acidic) and one basic amino group. The free amino group can readily undergo a condensation reaction, allowing fixation through an imine on tissue pretreated with glutaraldehyde or paraformaldehyde (PFA). This method ensures that suitable fluorescent labeling can be used to monitor the tissue area of interest.

To minimize autofluorescence and ensure successful fluorescence labeling, we recommend using the deep-red emitting dye ATTO-647N, which absorbs at 647 nm and emits light at approximately 665 nm (clean-red), well away from tissue autofluorescence. Combining two dyes, such as FITC-TRITC-dextran and ATTO488-ATTO647N-dextran at 4 and 10 kDa, can help distinguish different areas, such as the interstitial space and blood vessels. This approach avoids tissue autofluorescence and provides a very clean-red emission at 647 nm.

We provide ATTO647n-lysine-dextran as a standard product in our catalog that is fixable. However, we can always produce a non-fixable dextran labeled with the ATTO647n dye as a CSP (customer specific product).

The degree of fluorochrome binding typically remains consistent and is not dependent on the size of dextrans. We supply dextrans of various molecular sizes with remarkably similar degrees of labeling. Interestingly, different sizes of dextran exhibit nearly equivalent binding sites on glucose compared to the smaller ones. Dextrans generally behave as linear molecules rather than globular ones, so the entanglement of chains does not create sites with low accessibility for the dye. Consequently, the variations among the different sizes of dextrans we provide are minimal.

Yes. TdB Labs can conjugate dextran 10kDa with Oregon Green 488 dye and Fluorescein sodium as a customized product upon request.

The stability of specific products under certain biological conditions can be challenging to determine and there is limited data available. Further, the stability information provided is usually from our existing data files or literature search.

For the ARLD product, our data file states: “No prospective stability studies on Antonia Red Lysine Dextran have been performed yet. However, the structural properties of dextran and of the carbamide linkage of lysine to the dextran chain, as well as the covalent bond between lysine and Antonia Red, suggest high stability of the product.”

For the ATTO-LD product, there is no specific data file available. However, stability statements from ATTO-TEC about ATTO-protein conjugates, which should also apply to our ATTO-LD, indicate: “The conjugate should be stable at 4°C for several months. For long-term storage, divide the solution into small aliquots and freeze at -20°C. Avoid repeated freezing and thawing. Protect dye conjugates from light as much as possible.” We recommend referring to these guidelines, as we cannot provide more detailed stability information without additional data.

All high molecular dextran derivatives (≥150 kDa), with or without an attached dye, will be soluble when heated to 50 °C during continuous stirring for 30 min.

FITC inulin, and in some cases also blue dextrans, will be soluble when heated to 80 °C during continuous stirring.

DEAE- and CM-dextrans will be dissolved faster if you add the powder to water (not the other way around) during continuous stirring.

Yes, it is possible to have only one thiol per molecule. However, such a functionalization is random which means that to have an average molar ratio CMD/Thiol =1:1, we will risk having some of the molecules unmodified. In other words, the final product will be an ensemble of CMD and CMDSH molecules (where CMD4-SH corresponds to the thiol-modified CMD4). Also, it is worth mentioning that separating CMD4-SH from CMD4 is a difficult task.

Currently, we do not have the exact measurements but the degree of cross-linking in Polysucrose 20 and Polysucrose 40 lies between 5 and 15% (mol/mol).

No, we do not provide sterile products.

No, Pharma grade is not equivalent to GMP grade. Pharma grade products undergo an additional process step to reduce cross-contamination during production, and the product is tested for bioburdens and solvent residues. However, production is not conducted according to GMP standards because we lack the GMP license, which entails more stringent and detailed requirements for the lab environment, facilities, processes, etc.