Green fluorescent dye

Preparation of stock solution
1. Protein preparation:
Prepare protein (antibody) at a concentration of 1 mg/mL to achieve optimal labeling effect.
1) The protein solution's pH should be 8.5±0.5. In case the pH falls below 8.0, adjust the pH with 1 M NaHCO3.
2) The labeling efficiency will be significantly decreased if the protein content is less than 1 mg/mL. It is advised that the final protein concentration range be between 1 and 10 mg/mL in order to achieve the highest labeling efficiency.
3) To ensure optimal labeling efficacy, the protein needs to be in a clear buffer that contains primary amines (such Tris or glycine) and ammonium ions.
2. Preparation of the dye
Add the anhydrous DMSO to the FITC vial, shaking to make a 10 mM solution.
Note: FITC needs to be shielded from light and prepared freshly.
3. Determine how much dye is needed.
The amount of protein to be labeled determines how much FITC is needed for the labeling reaction. Approximately a mass ratio of 1:50 is ideal for FITC to protein.
For example, let's say that 1 mL of protein, 2 mg/mL of IgG (MW=150,000), and 1 mg of FITC mixed in 1 mL DMSO are the necessary labeling requirements. In this case, 40 μL of FITC is needed.
The following formula can be used to calculate:
Molar F/P=(MW/389)*(A495/195)/{[A280-(0.35*A495)]/E0.1%}=(A495*C)/[A280-(0.35*A495 )]
C=(MW*E0.1% 280)/(389*195)
*Note:
C represents a protein constant; MW is the molecular weight of a protein; 195 is FITC conjugate in pH=13, absorbance value E0.1% at 490 nm; (0.35×A495) is a correction factor based on FITC A280; E0.1% is the protein absorbance value (1.0 mg/mL) at 280 nm.

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Guidelines for usage.
1. Labeling reaction
1) Add 50 μL freshly prepared FITC to 1 mL of protein sample solution, gently shake and mix, then briefly centrifuge to collect the sample at the bottom of the reaction tube. Avoid vigorous mixing to prevent protein sample denaturation and inactivation.
2) Place the reaction tube in a dark place and incubate it gently at room temperature for 8 hours. Every 30 minutes, gently invert the reaction tube several times to fully mix the two reactants and improve labeling efficiency.
3) Add 5 M of NH4Cl to the final concentration of 50 mM, and terminate the reaction at 4 ℃ for 2 hours.
2. Protein purification and desalination
The following protocol takes the purification of dye protein conjugates using Sephadex G-25 column as an example.
1) Prepare Sephadex G-25 column according to the production instructions.
2) Load the reaction mixture into the top of the Sephadex G-25 column.
3) When the sample reaches below the surface of the top resin, immediately add PBS (pH 7.2-7.4).
4) Add more PBS (pH 7.2-7.4) to the required sample to complete column purification. The complex contains the required components of dye protein complexes.

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Safety Notes:
1. FITC is sensitive to light and humidity. Prepare FITC solution immediately and discard unused parts.
2. Low concentrations of sodium azide (≤ 3 mM or 0.02%) or thiomersal (≤ 0.02 mM or 0.01%) do not significantly interfere with protein labeling; But 20-50% glycerol will reduce labeling efficiency.
3. Avoid using buffers containing primary amines (such as Tris, glycine) or ammonium ions, as they compete with the labeled protein.
4. This product is limited to scientific research by professionals and shall not be used for clinical diagnosis or treatment, nor for food or medicine.
5. For your safety and health, please wear laboratory clothes and disposable gloves when operating.

Example 1: FITC can be used as a fluorescence probe for nanocomposites with green fluorescence.
Method: For nanocomposites linking.
1. Add nanocomposite to EDC and NHS (molar ratio: 1:5:5), and the pH is adjusted to activate the carboxyl groups.
2. Dissolve FITC in dimethyl sulfoxideab and add to the above mixture and shaked in the dark overnight.
3. The final FITC-labeled nanocomposite is obtained by lyophilization.
4. Use a probe-based confocal laser endoscopy (Cellvizio, Mauna Kea Technologies, France) for determination.

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Example 2: FITC can be used as a fluorescence probe for nanocomposites with green fluorescence.
Method: For nanocomposites linking.
1. Suspend nanocomposites (20 mg) in PBS (20 mL) with FITC (1 mg) and put the mixture stirred overnight in the dark.
2. Wash nanocomposites for five times with PBS to remove excess FITC.
3. Testing cells are seeded into a 6-well microplate at a density of 105/well and cultured overnight at 37°C in 5% CO2.
4. Change the culture medium with as-prepared medium containing nanocomposite-FITC. After coincubation for 1, 2, 4, and 8 h, testing cells are rinsed three times with PBS.
5. Use a confocal laser scanning microscopy for image.

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Example 3: FITC can be used as a fluorescence probe for linking to molecule glue with green fluorescence.

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Example 4: FITC can be used as a fluorescence probe for labeling lipophilic phytotoxin with green fluorescence.

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Example 5: FITC can be used as a fluorescence probe for labeling laccase with green fluorescence.
Method: For laccase labeling.
1. FITC (1 mg/mL) solution is prepared with dimethyl sulfoxide.
2. Dropwise add FITC into laccase solution (5 mg/mL), and stir the mixed solution at 4°C for 4 h.
3. Add NH4Cl aqueous solution (2 mL, 50 mM) into the mixed solution to stop the reaction.
4. Dialyze the solution in phosphate buffer (50 mM, pH 7) for 48 h at 4°C to remove excess FITC.
5. Use a confocal laser scanning microscopy (Leica SP8 STED 3X) for image.

[1]. Gao SG, et al. Phosphorylation of osteopontin has proapoptotic and proinflammatory effects on human knee osteoarthritis chondrocytes. Exp Ther Med. 2016 Nov;12(5):3488-3494. Epub 2016 Oct 5.
[2]. Gao SG, et al. Phosphorylation of osteopontin has proapoptotic and proinflammatory effects on human knee osteoarthritis chondrocytes. Exp Ther Med. 2016 Nov;12(5):3488-3494. Epub 2016 Oct 5.
[3]. Zhu X, et al. Ratiometric, visual, dual-signal fluorescent sensing and imaging of pH/copper ions in real samples based on carbon dots-fluorescein isothiocyanate composites. Talanta. 2017 Jan 1;162:65-71

C21H11NO5S

389.38

389.0358

C, 64.78; H, 2.85; N, 3.60; O, 20.54; S, 8.23

3326-32-7

Light yellow to brown solid

4

120.440

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

MHMNJMPURVTYEJ-UHFFFAOYSA-N

InChI=1S/C21H11NO5S/c23-12-2-5-16-18(8-12)26-19-9-13(24)3-6-17(19)21(16)15-4-1-11(22-10-28)7-14(15)20(25)27-21/h1-9,23-24H

3',6'-dihydroxy-5-isothiocyanato-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one

Fluorescein isothiocyanate isomer I Fluorescein 5-isothiocyanate FITC

2934.99.03.00

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)

DMSO : ~50 mg/mL (~128.41 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.34 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.08 mg/mL (5.34 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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