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FITC  (Synonyms: Fluorescein 5-isothiocyanate)

Cat. No.: HY-66019 Purity: 98.60%
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FITC (Fluorescein Isothiocyanate), is one of the green fluorescein derivatives widely used in biology. FITC has the characteristics of high absorptivity, excellent fluorescence quantum yield and good water solubility. The isothiocyanate group of FITC can be combined with amino, sulfhydryl, imidazole, tyrosyl, carbonyl and other groups on the protein, so as to achieve protein labeling including antibodies and lectins. In addition to its use as a protein marker, FITC can also be used as a fluorescent protein tracer to rapidly identify pathogens by labeling antibodies, or for microsequencing of proteins and peptides (HPLC). The maximum excitation wavelength of FITC is 494 nm. Once excited, it fluoresces yellow-green at a maximum emission wavelength of 520 nm.

For research use only. We do not sell to patients.

FITC Chemical Structure

FITC Chemical Structure

CAS No. : 3326-32-7

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Based on 40 publication(s) in Google Scholar

FITC Related Antibodies

Top Publications Citing Use of Products

35 Publications Citing Use of MCE FITC

IF

    FITC purchased from MedChemExpress. Usage Cited in: Adv Sci (Weinh). 2022 Jan 20;e2104344.  [Abstract]

    For detection of localization of WBC100 in cells, FITC labeled WBC100 (WBC100‐FITC) is used. HEK293T cells are transfected with the indicated plasmids for 48 h. Cells are treated with 1 μM WBC100‐FITC or FITC for 24 h.

    FITC purchased from MedChemExpress. Usage Cited in: J Nanobiotechnology. 2022 Feb 2;20(1):61.  [Abstract]

    To exam the ability of anti-PD-L1 antibodies (aPD-L1) nanovesicles to binding to tumor cells, B16F10 cells are cultured for 24 h. FITC-labeled aPD-L1 nanovesicles or nanovesicles are incubated with the cancer cells for 3 h.

    FITC purchased from MedChemExpress. Usage Cited in: Nat Commun. 2021 Nov 5;12(1):6426.  [Abstract]

    Cinnamate acetate (CIA) is labeled with FITC. Distribution of CIA/FITC in root cells of wild-type, ala7 mutant and complemented Arabidopsis seedlings. The seedlings are treated with CIA/FITC (5 μg/mL) and FM4-64 (8 μM) for 1 h, 2 h, and 8 h.

    FITC purchased from MedChemExpress. Usage Cited in: ACS Nano. 2020 Nov 24;14(11):14907-14918.  [Abstract]

    FITC-labeled MnO2/BPD are added at a concentration of 50 μg/mL and incubated for another 4 h to allow the nanoparticles to enter the cells. By conjugating FITC to MnO2/BPD, FL could be monitored at 488 nm, and the vessels could be observed at 660 nm by Evans Blue staining.

    FITC purchased from MedChemExpress. Usage Cited in: Chem Eng J. 2020, 127870.

    FITC (1 mg/mL) solution is prepared with dimethyl sulfoxide. Then FITC solution is added dropwise into laccase (5 mg/mL) solution, and the mixed solution is then stirred at 4 °C for 4 h. Laccase is labelled by FITC and exhibited green emissions.

    • Biological Activity

    • Protocol

    • Purity & Documentation

    • References

    • Customer Review

    Description

    FITC (Fluorescein Isothiocyanate), is one of the green fluorescein derivatives widely used in biology. FITC has the characteristics of high absorptivity, excellent fluorescence quantum yield and good water solubility. The isothiocyanate group of FITC can be combined with amino, sulfhydryl, imidazole, tyrosyl, carbonyl and other groups on the protein, so as to achieve protein labeling including antibodies and lectins. In addition to its use as a protein marker, FITC can also be used as a fluorescent protein tracer to rapidly identify pathogens by labeling antibodies, or for microsequencing of proteins and peptides (HPLC). The maximum excitation wavelength of FITC is 494 nm. Once excited, it fluoresces yellow-green at a maximum emission wavelength of 520 nm.

    In Vitro

    Protocol

    1.Protein Preparetion
    1) In order to obtain the best labeling effect, please prepare the protein (antibody) concentration as 2 mg/mL.
    2) The pH value of protein solution shall be 8.5±0.5. If the pH is lower than 8.0, 1m sodium bicarbonate shall be used for adjustment.
    3) If the protein concentration is lower than 2mg/ml, the labeling efficiency will be greatly reduced. In order to obtain the best labeling efficiency, it is recommended that the final protein concentration range is 2-10 mg/mL.
    4) The protein must be in the buffer without primary amine (such as Tris or glycine) and ammonium ion, otherwise the labeling efficiency will be affected.
    2.Dye Preparation
    Add anhydrous DMSO into the vial of FITC to make a 10 mM stock solution. Mix well by pipetting or vortex.
    3.Calculation of dye dosage
    The amount of FITC required for reaction depends on the amount of protein to be labeled, and the optimal molar ratio of FITC to protein is about 10.
    Example: assuming the required marker protein is 1 mL 2 mg/mL IgG (MW=150,000), use 1 mL DMSO dissolve 1 mg FITC, the required FITC volume is 40 μL.
    4.Run conjugation reaction
    1) A good volume of freshly prepared 10 mg/mL FITC is slowly added to 0.5 mL protein sample. In solution, gently shake to mix, then centrifuge briefly to collect the sample at the bottom of the reaction tube. Don't mix well to prevent protein samples from denaturation and inactivation.
    2) The reaction tubules were placed in a dark place and incubated gently at room temperature for 60 minutes at intervals.For 10-15 minutes, gently reverse the reaction tubules several times to fully mix the two reactants and raise the bar efficiency.
    5.Purify the conjugation
    The following protocol is an example of dye-protein conjugate purification by using a Sephadex G-25 column.
    1) Prepare Sephadex G-25 column according to the manufacture instruction.
    2) Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
    3) Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
    4) Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification.
    Combine the fractions that contain the desired dye-protein conjugate.

    Note
    1. FITC is sensitive to light and humidity. Immediately add FITC solution and discard the unused part.
    2. Low concentrations of sodium azide (≤3 mM or 0.02%) or thiomersal (≤0.02 mM or 0.01%) did not significantly interfere with protein labeling; However, 20-50% glycerol will reduce labeling efficiency.
    3. Avoid buffering with primary amines (e.g., Tris, glycine) or ammonium ions,It compete with labeled proteins.
    4. This product is only for scientific research by professionals, and shall not be used in clinical diagnosis or treatment, food or medicine.
    5. For your safety and health, please wear lab coat and disposable gloves.

    MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

    FITC Related Antibodies
    Molecular Weight

    389.38

    Formula

    C21H11NO5S
    CAS No.
    Appearance

    Solid

    Color

    Light yellow to brown

    Emission (Em)

    525
    Excitation (Ex)

    488
    SMILES

    O=C1OC2(C3=C(OC4=C2C=CC(O)=C4)C=C(O)C=C3)C5=C1C=C(N=C=S)C=C5
    Shipping

    Room temperature in continental US; may vary elsewhere.
    Storage

    -20°C, protect from light, stored under nitrogen

    *In solvent : -80°C, 6 months; -20°C, 1 month (protect from light, stored under nitrogen)

    Solvent & Solubility
    In Vitro: 

    DMSO : 50 mg/mL (128.41 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)

    H2O : < 0.1 mg/mL (insoluble)

    Preparing
    Stock Solutions
    Concentration Solvent Mass 1 mg 5 mg 10 mg
    1 mM 2.5682 mL 12.8409 mL 25.6819 mL
    5 mM 0.5136 mL 2.5682 mL 5.1364 mL
    View the Complete Stock Solution Preparation Table

    * Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
    Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light, stored under nitrogen). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

    • Molarity Calculator

    • Dilution Calculator

    Mass (g) = Concentration (mol/L) × Volume (L) × Molecular Weight (g/mol)

    Mass
    =
    Concentration
    ×
    Volume
    ×
    Molecular Weight *

    Concentration (start) × Volume (start) = Concentration (final) × Volume (final)

    This equation is commonly abbreviated as: C1V1 = C2V2

    Concentration (start)

    C1

    ×
    Volume (start)

    V1

    =
    Concentration (final)

    C2

    ×
    Volume (final)

    V2

    In Vivo:

    Select the appropriate dissolution method based on your experimental animal and administration route.

    For the following dissolution methods, please ensure to first prepare a clear stock solution using an In Vitro approach and then sequentially add co-solvents:
    To ensure reliable experimental results, the clarified stock solution can be appropriately stored based on storage conditions. As for the working solution for in vivo experiments, it is recommended to prepare freshly and use it on the same day.
    The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.

    • Protocol 1

      Add each solvent one by one:  10% DMSO    40% PEG300    5% Tween-80    45% Saline

      Solubility: ≥ 2.08 mg/mL (5.34 mM); Clear solution

      This protocol yields a clear solution of ≥ 2.08 mg/mL (saturation unknown).

      Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (20.8 mg/mL) to 400 μL PEG300, and mix evenly; then add 50 μL Tween-80 and mix evenly; then add 450 μL Saline to adjust the volume to 1 mL.

      Preparation of Saline: Dissolve 0.9 g sodium chloride in ddH₂O and dilute to 100 mL to obtain a clear Saline solution.
    • Protocol 2

      Add each solvent one by one:  10% DMSO    90% (20% SBE-β-CD in Saline)

      Solubility: 2.08 mg/mL (5.34 mM); Suspended solution; Need ultrasonic

      This protocol yields a suspended solution of 2.08 mg/mL. Suspended solution can be used for oral and intraperitoneal injection.

      Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (20.8 mg/mL) to 900 μL 20% SBE-β-CD in Saline, and mix evenly.

      Preparation of 20% SBE-β-CD in Saline (4°C, storage for one week): 2 g SBE-β-CD powder is dissolved in 10 mL Saline, completely dissolve until clear.
    In Vivo Dissolution Calculator
    Please enter the basic information of animal experiments:

    Dosage

    mg/kg

    Animal weight
    (per animal)

    g

    Dosing volume
    (per animal)

    μL

    Number of animals

    Recommended: Prepare an additional quantity of animals to account for potential losses during experiments.
    Please enter your animal formula composition:
    %
    DMSO +
    % 
    +
    %
    Tween-80 +
    %
    Saline
    Recommended: Keep the proportion of DMSO in working solution below 2% if your animal is weak.
    The co-solvents required include: DMSO, . All of co-solvents are available by MedChemExpress (MCE). , Tween 80. All of co-solvents are available by MedChemExpress (MCE).
    Calculation results:
    Working solution concentration: mg/mL
    Method for preparing stock solution: mg drug dissolved in μL  DMSO (Stock solution concentration: mg/mL).

    *In solvent : -80°C, 6 months; -20°C, 1 month (protect from light, stored under nitrogen)

    The concentration of the stock solution you require exceeds the measured solubility. The following solution is for reference only. If necessary, please contact MedChemExpress (MCE).
    Method for preparing in vivo working solution for animal experiments: Take μL DMSO stock solution, add μL . μL , mix evenly, next add μL Tween 80, mix evenly, then add μL Saline.
     If the continuous dosing period exceeds half a month, please choose this protocol carefully.
    Please ensure that the stock solution in the first step is dissolved to a clear state, and add co-solvents in sequence. You can use ultrasonic heating (ultrasonic cleaner, recommended frequency 20-40 kHz), vortexing, etc. to assist dissolution.
    Purity & Documentation

    Purity: 98.60%

    COA (256 KB) RP-HPLC (244 KB) MS (68 KB)

    Handling Instructions (2659 KB)
    Dyeing Example
    References
    Cell Assay
    [1]

    The frequency of chondrocyte apoptosis is measured by flow cytometry with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide. A total of 1×104 treated chondrocytes are collected from each group, washed in cold PBS and incubated with Annexin V-FITC and PI at room temperature for 15 min in the dark on ice. These samples are then analyzed using a fluorescence-activated cell sorter. Cell Quest software is used to analyze the percentage of apoptosis. All tests are repeated in triplicate.

    MCE has not independently confirmed the accuracy of these methods. They are for reference only.
    References

    Complete Stock Solution Preparation Table

    * Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
    Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light, stored under nitrogen). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

    Optional Solvent Concentration Solvent Mass 1 mg 5 mg 10 mg 25 mg
    DMSO 1 mM 2.5682 mL 12.8409 mL 25.6819 mL 64.2046 mL
    5 mM 0.5136 mL 2.5682 mL 5.1364 mL 12.8409 mL
    10 mM 0.2568 mL 1.2841 mL 2.5682 mL 6.4205 mL
    15 mM 0.1712 mL 0.8561 mL 1.7121 mL 4.2803 mL
    20 mM 0.1284 mL 0.6420 mL 1.2841 mL 3.2102 mL
    25 mM 0.1027 mL 0.5136 mL 1.0273 mL 2.5682 mL
    30 mM 0.0856 mL 0.4280 mL 0.8561 mL 2.1402 mL
    40 mM 0.0642 mL 0.3210 mL 0.6420 mL 1.6051 mL
    50 mM 0.0514 mL 0.2568 mL 0.5136 mL 1.2841 mL
    60 mM 0.0428 mL 0.2140 mL 0.4280 mL 1.0701 mL
    80 mM 0.0321 mL 0.1605 mL 0.3210 mL 0.8026 mL
    100 mM 0.0257 mL 0.1284 mL 0.2568 mL 0.6420 mL
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    FITC Related Classifications

    Help & FAQs
    • Do most proteins show cross-species activity?

      Species cross-reactivity must be investigated individually for each product. Many human cytokines will produce a nice response in mouse cell lines, and many mouse proteins will show activity on human cells. Other proteins may have a lower specific activity when used in the opposite species.

    Keywords:

    FITC3326-32-7Fluorescein 5-isothiocyanateFluorescent DyeInhibitorinhibitorinhibit

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