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Fluorescein Isothiocyanate-Dextran, 4 kDa
Catalog # 4013
For Research Use Only - Not Human or Therapeutic Use

DESCRIPTION:

Fluorescein isothiocyanate (FITC) labeled dextran

APPLICATION:

Use to assess the permeability of semi-permeable membranes either in vivo or in vitro (1-4).
NOTE: FITC-dextran can be used simultaneously with TRITC-dextran (Cat # 4014) as
fluorescence occurs at different wavelengths.

QUANTITY:

5 ml

FORM:

25 mg/ml solution in 0.05M phosphate buffered saline

MOLECULAR WEIGHT:

4 kDa

FLUORESCENCE:

Excitation: 490 nm, Emission: 520 nm

STORAGE:

4⁰C in the dark

STABILITY:

1 yearIN VIVO PROTOCOL:
1.

Fast mice 4 hours before oral feeding and for the duration of the experiment.

2.

Feed 20 ml/kg by oral gavage.

3.

Maintain fasting conditions and wait 3 hours (may vary depending on individual animals).

4.

Collect blood by retro-orbital bleeding, then spin and collect plasma. Dilute plasma 1:2 (ormore) with PBS.
NOTE 1: Protein in the samples may interfere with and reduce the fluorescence intensity(FI). Therefore, in order to accurately determine FITC-dextran permeability, the plasma toPBS ratio must be consistent throughout all the samples.
NOTE 2: Chondrex, Inc. recommends making a standard curve from serial dilutions of thestock FITC-dextran for qualitative studies.

5.

Transfer 50 or 100 μl of diluted standards and samples to a black 96-well plate and readin a fluorescence reader.
Settings for Reading: Excitation: 490 nm/Emission: 520 nm
Wavelength Bandwidth (Excitation and Emission): 9 nm
Gain: Auto or set 80,000 equivalent to 12.5 μg/ml© 2020 Chondrex, Inc. All Rights Reserved, 4013 3.016928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

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IN VITRO PROTOCOL:

A protocol for in vitro studies will vary due to the types of cultured cells, culture systems, purposeof study, etc. Please contact support@chondrex.com for more information about optimization.

PREPARING STANDARDS:

A standard range of 12.5 to 0.2 μg/ml is recommended. To prepare standard dilutions, use adiluent with the same ratio of normal mouse plasma to PBS as the samples. For example, ifusing a 1:2 dilution for sample dilution (in vivo protocol, Step 4), prepare 33% normal mouseplasma in 0.05M phosphate buffered saline (300 μl plasma with 600 μl PBS) as a diluent.
1.

10 μl of 25 mg/ml FITC-Dextran with 990 μl of PBS (250 μg/ml).

2.

12.5 μl of the diluted FITC-Dextran with 237.5 μl of 33% normal mouse plasma in 0.05M
phosphate buffered saline (12.5 μg/ml).

3.

Mix 125 μl of the 12.5 μg/ml solution with an equal volume of 33% mouse normal plasma(6.3 μg/ml).

4.

Repeat 5 times for the 3.1, 1.6, 0.8, 0.4, and 0.2 μg/ml standard solutions.

5.

Transfer 50 or 100 μl of diluted standards and samples to a black 96-well plate and readon a fluorescence reader.

6.

Subtract the FI blank values (33% normal mouse plasma in 0.05M phosphate bufferedsaline) from the FI values of the standards and samples.

7.

Plot the FI values of the standards against the μg/ml of the FITC-Dextran standards. Usinga log/log plot will linearize the data (Figure 1).

Figure 1. A Typical Standard Curve for FITC-Dextran(µg/ml)
12.5
6.25
3.13
1.56
0.78
0.39
0.20
33% Serum/PBS

NOTES:FI Values
76281
39052
20389
10414
5471
2903
1632
431

Corrected
75850
38621
19958
9983
5040
2472
1201
0

N/A

REFERENCES:
1.

M. Vijay-Kumar, C. Sanders, R. Taylor, A. Kumar, J. Aitken, et al., Deletion of TLR5 resultsin spontaneous colitis in mice. J Clin Invest 117, 3909-21 (2007).

2.

Q. Wang, C. Fang, P. Hasselgren, Intestinal permeability is reduced and IL-10 levels areincreased in septic IL-6 knockout mice. Am J Physiol Regul Integr Comp Physiol 281,R1013-23 (2001).

3.

3. W. Huang et al., HMGB1 increases permeability of the endothelial cell monolayer via
RAGE and Src family tyrosine kinase pathways. Inflammation 35, 350-362 (2012).

© 2020 Chondrex, Inc. All Rights Reserved, 4013 3.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com