Fluorescent Dye

Fluorescein isothiocyanate (FITC) dextran fluorochrome (Ex=495 nm; Em=525 nm) is known as FITC-Dextran (MW 500000). In order to investigate the early and late phases of cell sealing and to identify heat shock-induced cellular damage, FITC-Dextran (MW 500000) can be employed as a marker. For cell permeability investigations, such as blood-brain barrier permeability and assessment of the extent of blood-brain barrier disruption, FITC-Dextran (MW 500000) is utilized. Storage: Keep out of direct sunlight.

Standard Procedure
(Below is our suggested protocol, which should be adapted as needed for your specific application.)
Intestinal Barrier Function Assay [5]
1. Fasting Treatment
Mice were fasted for 4 hours prior to the experiment.

2. FITC-Dextran (MW 70,000) Administration
Oral gavage was performed using FITC-Dextran (0.6 mg/g body weight).

3. Fluorescence Measurement
Serum fluorescence intensity was measured within 4 hours post-administration.

Detection parameters:
Excitation (Ex): 490 nm
Emission (Em): 520 nm
Key Notes:
This assay evaluates intestinal permeability by measuring FITC-Dextran leakage into circulation.
Higher fluorescence indicates compromised gut barrier integrity.
Ensure proper fasting control to avoid interference from food digestion.

Culturing of Cells and Loading with FITC-Dextran (See Note 2 ) [3]
1. Human fibroblasts cultured in cell culture medium are incubated in humidified air with 5% CO2 at 37 °C and subcultured once a week (see Note 3 ).
2. Trypsinize, count and seed cells at a density of 9000 cells/cm2 (see Note 4 ) in a ∅ 35 mm cell culture dish. We recommend using at least triplicates for samples and a standard curve consisting of five pH-values (e.g., pH 4.05, 4.5, 5.0, 5.5, and 6) (see Note 5 ). Also include an unstained sample.
3. Make a solution of 1 mg/ml FITC-Dextran in cell culture medium. Filter-sterilize the solution through a hydrophilic polyethersulfone membrane by using a syringe-driven filter unit with 0.22 μM pore size.
4. Prepare cell culture medium containing FITC-Dextran at a final concentration of 0.1 mg/ml (see Note 6 ).
5. Aspire the cell culture media from the cells and add 1 ml of FITC-Dextran containing medium and incubate for 3 days (see Note 7 ) at 37 °C with 5% of CO2 in air.

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Preparations for Measurement of Lysosomal pH (See Note 7 )[3]
1. Chase the FITC-Dextran to lysosomes by aspirating the media and add fresh cell culture medium, incubate cells for 2 h (see Note 8 ). If desired, cells can be examined in a fluorescence microscope to verify FITC-Dextran accumulation in a punctate pattern corresponding to lysosomes (Fig. 2). During this period, also expose cells to LMP-inducers or inhibitors, if desired (see Note 9 ).
2. After the chase period, detach cells by trypsination and transfer to tubes.
3. Centrifuge at 300 × g for 5 min and remove the medium by aspiration.
4. Wash the cells in 1 ml of PBS (room temperature). Recentrifuge the tubes at 300 × g for 5 min.
5. Pour off the PBS and place the samples on ice.
6. Prepare an appropriate volume Britton-Robinson buffer of each pH (0.5 ml/standard sample). Add sodium azide and 2-deoxyglucose to a final concentration of 50 mM and nigericin to a final concentration of 10 μM (see Note 10 ). Keep the buffers on ice.

[1]. Fluorescein isothiocyanate-dextran can track apoptosis and necrosis induced by heat shock of peripheral blood mononuclear cells and HeLa cells. Open Biological Sciences Journal, 2015, 1(1).

[2]. Fluorescein Isothiocyanate (FITC)-Dextran Extravasation as a Measure of Blood-Brain Barrier Permeability. Curr Protoc Neurosci. 2017 Apr 10;79:9.58.1-9.58.15.

[3]. Analysis of Lysosomal pH by Flow Cytometry Using FITC-Dextran Loaded Cells. Methods Mol Biol. 2017;1594:179-189.

[4]. Cdc42 activates paracellular transport in polarised submandibular gland cells. Arch Oral Biol. 2021 Dec;132:105276.

[5]. ACE2 contributes to the maintenance of mouse epithelial barrier function. Biochem Biophys Res Commun. 2020 Dec 17;533(4):1276-1282.

Critical Parameters [2]
1. It is important to use a correct dose of ketamine/xylazine cocktail for anesthesia. The dose should be sufficient for the rat to remain at a surgical plane of anesthesia without being lethal. For good perfusion to occur the heart should be beating at regular intervals when the heparin is injected into the left ventricle. The heparin injection is given in order to prevent platelet aggregation within the blood vessels and to provide a free flow of blood without any back pressure during perfusion, thereby minimizing rupture of small blood vessels and capillaries.
2. The flow of the perfusion solution needs to be maintained at a rate that is not too high as i
t would increase pressure on small capillaries causing them to rupture. On the other hand pressure has to be maintained sufficiently to perfuse the smallest diameter capillaries. 3. The incision made in the right atrium for blood outflow has to be sufficiently large (~0.5 cm) for the blood to empty freely without building back pressure.
4. The concentration of FITC-Dextran solution used in perfusion should be high enough to overcome the dilution by blood in the vessels during perfusion. The optimal concentration of FITC-dextran to use in perfusion should be empirically determined by the experimenter based on the weight of the animal.
5. It is important to ensure that FITC-dextran powder is completely solubilized prior to perfusion and is cold.
6. Rats must be decapitated and the brain removed immediately after perfusion with FITC-Dextran is completed. This would minimize artificial leakage of FITC-dextran from the vessels in the absence of active perfusion by the pump. 7. Cryoprotection of the brain is essential for preventing sudden osmotic changes in cells during flash freezing that would burst the cell wall, damage tissue, and contribute to artificial leakage of FITC-dextran.
8. Do not immerse brains in 2-methylbutane for longer than 5 min as it causes fracturing of the tissue.
9. Using subbed slides for mounting tissue sections helps the tissue adhere to and flatten on the glass slide. Additionally, it prevents the sections from lifting off the slide when incubating in DRAQ5.
10. It is essential to equilibrate the brains to the temperature of the cryostat for at least 2 hr. Frequently, the outside of the brain is at a different temperature compared to the inner regions and this interferes with obtaining consistent slice thickness and also affects the quality of the section.
11. The temperature at which the sections are sliced is important. Sectioning when it is too warm will make the tissue clump and stick to itself. In contrast, if the cryostat temperature is too cold, the tissue will crack and roll up tightly and make it difficult to unroll without causing damage to the tissue. Appropriate temperature setting of the cryostat depends on ambient temperature and humidity, and should be determined prior to sectioning of the area of interest by assessing the quality of sections from a region of the brain that is not of interest.
12. After sectioning, the tissue slices should be carefully and gently transferred from the cryostat blade using a thin brush and placed on the slide, as the blood vessels can break due to mechanical agitation and FITC can leak out of the capillaries.
13. Low-light conditions should be maintained when working with the FITC-Dextran perfused brain tissue to minimize the degradation of fluorescence due to quenching by ambient bright light. Low ambient light and covering the brains and tissue with foil help maintain fluorescence.
14. Slide mounted tissue sections need to be completely rehydrated in PBS (with or without DRAQ5) prior to Fluoromount-G application and coverslipping. Fluoromount-G is a mounting media that works well on wet tissue but its application to dry dehydrated sections forms numerous small air bubbles during setting of the mounting media.
15. Do not wash the section after incubation with DRAQ5 as it could wash out FITC-Dextran and decrease the DRAQ5 signal making it difficult to identify the correct focal plane during imaging.
16. Both control and treated groups need to be imaged in every session. This would take into account any day-to-day variability or unintended changes in the confocal microscope and treatment of the slides.
17. Same brain areas/regions should be imaged in control and experimental groups in one imaging session rather than imaging all the brain regions of one group followed by a different group.
18. Once the imaging parameters are established, the investigator performing the analyses should be blind to the treatment conditions in order to eliminate introduction of bias to imaging measures.

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Troubleshooting
1. Too dark or no visible capillaries in a control rat can be due to poor perfusion
a. Liver clearing can be used as an indicator of perfusion. The liver should begin to clear out in 5 – 10 s after starting perfusion. If this does not happen, readjust the position of the 16 G needle in the ventricle making sure that the beveled tip is not against the wall of the heart or septum.
b. Increase the volume of FITC-Dextran perfused. The amount of FITC-dextran suggested (12 mL) is for rats weighing between 250–300 g. Heavier rats may require a greater volume of perfused FITC-dextran to fill the small capillaries in the brain.
c. An increase in the pump flow rate could also be tried, however there is a concern that higher speeds and therefore, higher pressure, could cause brain capillaries to rupture and leak FITC-Dextran, leading to false positives for BBB disruption. Keep the pump rate consistent for all control and experimental groups so that changes in the rate of the pump do not unintentionally cause variability in BBB disruption.

2. Bright fluorescent speckles all over the section and slides during imaging
a. Too much disruption of the tissue section when slicing or placing onto subbed slides can rupture blood vessels and capillaries causing FITC-Dextran to leak out.
b. Kimwipes or other lab tissues can leave behind specks and debris that fluoresce at different wavelengths of light and should not be used for cleaning slides. A soft microfiber cloth is a better alternative for wiping slides clean.
c. The freezing process could also lead to bright fluorescent specks over the tissue. Incubation in the 2-methylbutane for longer than 5 min could cause tissue dehydration and rupture of cell membranes.

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The blood-brain barrier (BBB) is formed in part by vascular endothelial cells that constitute the capillaries and microvessels of the brain. The function of this barrier is to maintain homeostasis within the brain microenvironment and buffer the brain from changes in the periphery. A dysfunction of the BBB would permit circulating molecules and pathogens typically restricted to the periphery to enter the brain and interfere with normal brain function. As increased permeability of the BBB is associated with several neuropathologies, it is important to have a reliable and sensitive method that determines BBB permeability and the degree of BBB disruption. A detailed protocol is presented for assessing the integrity of the BBB by transcardial perfusion of a 10,000 Da FITC-labeled dextran molecule and its visualization to determine the degree of extravasation from brain microvessels. [2]

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The acidic environment of the lysosomal lumen provides an optimal milieu for the acid hydrolases and is also essential for fusion/fission of endo-lysosomal compartments and sorting of cargo. Evidence suggests that maintaining lysosomal acidity is essential to avoid disease. In this chapter, we describe a protocol for analyzing the lysosomal pH in cultured cells using the fluorescent probe fluorescein isothiocyanate (FITC)-dextran together with a dual-emission ratiometric technique suitable for flow cytometry. Fluorescence-labeled dextran is endocytosed and accumulated in the lysosomal compartment. FITC shows a pH-dependent variation in fluorescence when analyzed at maximum emission wavelength and no variation when analyzing at the isosbestic point, thereby the ratio can be used to determine the lysosomal pH. A standard curve is obtained by equilibrating intralysosomal pH with extracellular pH using the ionophore nigericin. The protocol also includes information regarding procedures to induce lysosomal alkalinization and lysosomal membrane permeabilization. [3]

C21H13NO6S.XUNSPECIFIED

60842-46-8

Yellow to orange solid powder

C1=C(O)C=CC2C3(OC(=O)C4=CC=CC=C34)C3=C(C=C(ONC(=O)S)C=C3)OC1=2

FITC-Dextran (MW 70000)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~50 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)