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Tetramethylrhodamine Isothiocyanate-Dextran, 70 kDa
Catalog # 4014
For Research Use Only - Not Human or Therapeutic Use

DESCRIPTION:

Tetramethylrhodamine isothiocyanate (TRITC) labeled dextran

APPLICATION:

Use to assess the permeability of semi-permeable membranes either in vivo or in vitro (1-4).
NOTE: TRITC-dextran can be used simultaneously with FITC-dextran (Cat # 4009 or 4013) as
fluorescence occurs at different wavelengths.

QUANTITY:

5 ml

FORM:

25 mg/ml solution in 0.05M phosphate buffered saline

MOLECULAR WEIGHT:

70 kDaFLUORESCENCE:

Excitation: 550 nm, Emission: 572 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 TRITC-dextran permeability, the plasma
to PBS ratio must be consistent throughout all the samples.
NOTE 2: Chondrex, Inc. recommends making a standard curve from serial dilutions of thestock TRITC-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: 550 nm/Emission: 572 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, 4014 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

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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, if
using 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 TRITC-Dextran with 990 μl of PBS (250 μg/ml).

2.

12.5 μl of the diluted TRITC-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 TRITC-Dextran standards.
Using a log/log plot will linearize the data (Figure 1).

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

NOTES:FI Values
77804
41765
25476
16829
12706
9525
8290
7044

Corrected
70760
34721
18432
9785
5662
2481
1246
0

N/A

REFERENCES:
1.

D. Fernández-López, J. Faustino, R. Daneman, L. Zhou, S. Lee, et al., Blood-brain
barrier permeability is increased after acute adult stroke but not neonatal stroke in therat. J Neurosci 32, 9588-600 (2012).

2.

J. Kim, J. Lindsey, N. Wang, R. Weinreb, Increased human scleral permeability withprostaglandin exposure. Invest Ophthalmol Vis Sci 42, 1514-21 (2001).

3.

D. Pink, W. Schulte, M. Parseghian, A. Zijlstra, J. Lewis, Real-time visualization andquantitation of vascular permeability in vivo: implications for drug delivery. PLoS One 7,e33760 (2012).

4.

R. Sajja, S. Prasad, L. Cucullo, Impact of altered glycaemia on blood-brain barrier
endothelium: an in vitro study using the hCMEC/D3 cell line. Fluids Barriers CNS 11, 8(2014).

© 2020 Chondrex, Inc. All Rights Reserved, 4014 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