Fluorescent Dye

Fluorescein isothiocyanate (FITC) dextran fluorochrome (Ex=495 nm; Em=525 nm) is known as FITC-Dextran (MW 2000000) (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 2000000) (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 2000000) (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 2000000) (MW 70,000) Administration
Oral gavage was performed using FITC-Dextran (MW 2000000) (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 (MW 2000000) 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 (MW 2000000) (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 (MW 2000000) 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 (MW 2000000) 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 (MW 2000000) 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 (MW 2000000) 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 (MW 2000000) 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 (MW 2000000) 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.

Key parameters [2]
1. It is crucial to use the correct dose of ketamine/xylazine mixture for anesthesia. The dose should be sufficient to maintain the surgical anesthesia depth in rats without causing death. To ensure good perfusion, the heart should maintain a regular beating while heparin is injected into the left ventricle. Heparin is injected to prevent platelet aggregation in the blood vessels and to provide free flow of blood without back pressure during perfusion, thereby minimizing the rupture of small blood vessels and capillaries.
2. The flow rate of the perfusion fluid needs to be kept at a level that is not too high, as excessive flow rate will increase the pressure in small capillaries, leading to their rupture. On the other hand, sufficient pressure must be maintained to perfuse the smallest diameter capillaries. 3. The incision in the right atrium for blood outflow must be large enough (about 0.5 cm) so that blood can flow out freely without back pressure. 4. The concentration of the FITC-glucan (molecular weight 2,000,000) solution used in perfusion should be high enough to overcome the dilution of blood in the blood vessels during perfusion. The optimal concentration of FITC-glucan (molecular weight 2,000,000) used for perfusion should be empirically determined by the experimenter based on the animal's body weight. 5. It is essential to ensure that the FITC-glucan (molecular weight 2,000,000) powder is completely dissolved and cryogenic before perfusion. 6. After perfusion with FITC-glucan (molecular weight 2,000,000), the rat must be immediately decapitated and the brain tissue removed. This minimizes artificial leakage of FITC-glucan (molecular weight 2,000,000) from the blood vessels without active perfusion by the pump. 7. Cryoprotection of the brain is crucial to prevent sudden changes in intracellular osmotic pressure during rapid freezing, which can lead to cell wall rupture, tissue damage, and artificial leakage of FITC-glucan (molecular weight 2,000,000). 8. Do not immerse the brain in 2-methylbutane for more than 5 minutes, as this can cause tissue rupture. 9. Using pre-treated slides to fix tissue sections helps the tissue adhere to the slide and flatten it. It also prevents sections from detaching from the slide during incubation in DRAQ5. 10. The brain must be equilibrated with the cryostat temperature for at least 2 hours. Typically, the external temperature of the brain differs from the internal temperature, which affects the consistency of section thickness and the quality of the sections. 11. Sectioning temperature is crucial. Excessive temperature can cause tissue to clump and adhere together. Conversely, if the cryostat temperature is too low, the tissue will crack and curl tightly, making it difficult to unfold without causing tissue damage. The appropriate cryostat temperature setting depends on the ambient temperature and humidity and should be determined by evaluating the quality of sections from non-target brain regions before sectioning the target area. 12. After sectioning, the tissue sections should be carefully and gently transferred from the cryostat blade to the slide using a fine-bristled brush, as mechanical shaking can cause blood vessels to rupture, and FITC may leak from capillaries. 13. When using brain tissue perfused with FITC-glucan (molecular weight 2,000,000), low light conditions should be maintained to minimize fluorescence decay caused by ambient light quenching. Low light conditions and covering the brain tissue with aluminum foil help maintain fluorescence. 14. Tissue sections on slides need to be fully rehydrated with PBS (with or without DRAQ5) before applying Fluoromount-G mounting medium and coverslips. Fluoromount-G is a mounting medium suitable for wet tissues, but when used on dried and dehydrated sections, it can generate numerous small air bubbles during the curing process. 15. Do not wash sections after incubation with DRAQ5, as this may wash away FITC-glucan (molecular weight 2,000,000), reducing the DRAQ5 signal and making it difficult to determine the correct focal plane during imaging. 16. Imaging of both the control and treatment groups should be performed for each experiment. This will account for any daily variability or unexpected changes in confocal microscopy and slide handling. 17. The control and experimental groups should be imaged in the same brain region/area in a single imaging session, rather than imaging all brain regions of one group first and then the other. 18. Once imaging parameters are determined, the researchers performing the analysis should be unaware of the processing conditions to avoid introducing bias into the imaging measurements. Troubleshooting 1. Dark images or indistinct capillaries in the control group rats may be due to poor perfusion. a. Liver clearance can be used as an indicator of perfusion. After perfusion begins, the liver should begin to clear within 5-10 seconds. If this does not occur, readjust the position of the 16G needle in the ventricle, ensuring the bevel tip does not touch the heart wall or interventricular septum. bt. Increase the volume of FITC-glucan (molecular weight 2,000,000) perfused. The recommended dosage of FITC-glucan (molecular weight 2,000,000) (12 mL) is suitable for rats weighing between 250-300 grams. Heavier mice may require a larger volume of FITC-glucan (molecular weight 2,000,000) to fill the small capillaries in their brains. CT scans could also attempt to increase the pump flow rate, but there are concerns that higher flow rates (and thus higher pressures) could cause capillary rupture and leakage of FITC-glucan, leading to false-positive results of blood-brain barrier disruption. Maintaining consistent pump rates across all control and experimental groups is crucial to avoid unexpected variations in the degree of blood-brain barrier disruption caused by pump rate changes. 2. Bright fluorescent spots on sections and slides during imaging: a. Excessive disturbance of tissue sections during sectioning or placement on slides can cause rupture of blood vessels and capillaries, resulting in FITC-glucan leakage. b. Kimberly-Clark wipes or other laboratory tissues may leave spots and debris that fluoresce at different wavelengths and should therefore not be used to clean slides. Wiping the slides with a soft microfiber cloth is a better option.
c. The freezing process may also result in bright fluorescent spots on the tissue. Incubation in 2-methylbutane for more than 5 minutes may cause tissue dehydration and cell membrane rupture.

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The blood-brain barrier (BBB) is partially formed by vascular endothelial cells that make up the brain's capillaries and microvessels. This barrier functions to maintain the homeostasis of the brain microenvironment and buffer the brain from changes in the peripheral environment. BBB dysfunction allows circulating molecules and pathogens that are normally confined to the periphery to enter the brain, interfering with normal brain function. Since increased BBB permeability is associated with a variety of neuropathologies, having a reliable and sensitive method to determine the permeability and extent of BBB disruption is crucial. This article details a protocol for assessing BBB integrity by perfused 10,000 Da FITC-labeled dextran molecules via the heart and visualized to determine the extent of extravasation from brain microvessels. [2] The acidic environment of the lysosomal cavity provides the optimal activity environment for acidic hydrolases and is also crucial for the fusion/division of endosome-lysosomal compartments and the sorting of cargo. There is evidence that maintaining the acidity of lysosomes is essential for disease prevention. This chapter introduces a protocol for analyzing the pH of lysosomes in cultured cells using the fluorescent probe fluorescein isothiocyanate (FITC)-dextran and a dual emission ratio technique suitable for flow cytometry. The fluorescently labeled dextran is endocytosed and accumulates in the lysosomal compartments. When analyzed at the maximum emission wavelength, the fluorescence intensity of FITC changes with pH; when analyzed at the isoabsorption point, the fluorescence intensity does not change. Therefore, this ratio can be used to determine the pH of lysosomes. A standard curve can be obtained by balancing the pH inside the lysosome and the extracellular pH using the ionocarrier nigericin. The protocol also includes information on methods for inducing lysosomal alkalization and lysosomal membrane permeability. [3]

C21H13NO6S.XUNSPECIFIED

2000000

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 2000000)

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.)