Fibronectin is a major protein found in
human plasma that plays an important role in wound healing and
embryonic development. The important property of fibronectin to
mediate cell attachment is used in vitro to create more
physiologically relevant surfaces for culturing cells. Addition of
fibronectin is especially beneficial to promote cell attachment in
serum-free media.
Fibronectin is is purified by affinity chromatography. It is then lyophilized (from 2.5 ml of
buffer (150 mM NaCl, 10 mM sodium phosphate pH 7.5) to ensure
stability in storage and during shipping. Purity 95% (SDS-PAGE - double band 220 kDa).
Source: Human plasma. Donors tested negative for Hepatitis A and B, HCV, HIV1, HIV2, Parvovirus B19
Fibronectin is shipped at ambient
temperature. Upon arrival store dry at –20°C or lower. Lyophilized
product is moisture-sensitive. Reconstituted and sterile-filtered
product can be stored for 1 month at +2...8°C
Reconstitution of lyophilized
fibronectin is done with 5 ml of warm (37°C) PBS . Incubate at 37°C
for 3 hours, occasionally inverting the vial. Do not vortex.
Reconstituted solution contains 5 mg of soluble fibronectin and a
minor quantity of insoluble fibronectin aggregate. Filter slowly
through prewetted 0.2 µm membrane filter. Concentration 1 mg/ml of fibronectin
corresponds to A280 - 1.3
Amount 5 mg
Applications
- Attachment of circulating angiogenic cell CACs (1)
- Coating of polymer coverslips with fibronectin for attachment of HEK293.123 cells (2)
- Attachment of pathogenic Escherichia coli and Salmonella through bacterial curli protein CsgA to fibronectin (3)
- Adhesion of human dermal fibroblasts to fibronectin (4)
- Fibronectin was used as a cell-adhesive factor on microstructured single-cell arrays of A549 and Huh7 cells (5)
- Culture of human induced pluripotent stem cell-derived cardiomyocytes hiPSC-CMs on fibronectin-coated glass dishes (6)
- Attachment and culture of MDCK cells on fibronectin-micropatterned surfaces (7)
- Use of fibronectin for PAA gel cell culture of NIH-3T3 cells (8)
- Cell adhesion for single-cell arrays of A549, HuH7, and MDA-MB-436 cell lines (9)
- Cell adhesion protein for adhesion and growth of normal human dermal fibroplasts (NHDF) to create highly selective nanostructured surfaces (nanosphere lithography NSL) (10)
- HUVEC cell culture (11)
- Surface coating of a corneal implant made of collagen hydrogel to enhance the attachment and growth of human corneal epithelial cells, microcontact printing with fibronectin to promote cell adhesion and cell attachment, biointeractive scaffold to mimic the extracellular matrix (ECM) for regenerative medicine, lithographc patterning for cell attachment of corneal implants (12)
- Cell culture on fibronectin-coated islands of pre-determined size and shape created by soft lithography on NHS-functionalized acrylamide (13)
- Fibronectin was used for creating a pattern on substrate that was used for attachment and growth of induced pluripotent stem cell (iPSC)-derived cardiomyocytes (14)
- Microcontact fibronectin printing on cytophobic polyacrylamide gels. Fibronectin forms a stable cell-adherent pattern for OS-HEK cell line (15)
- Attachment of human dermal fibroblasts (16, 17)
Citations
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Kat, Hendrik Gremmels, Marianne C. Verhaar, Frank J. M. Broekmans, and Felicia
Yarde, “Early Vascular Damage in Young Women with DM-1 and Its Relation to
Anti-Müllerian Hormone: A Cross-Sectional Study,” International Journal of Endocrinology,
vol. 2016, Article ID 1487051, 7 pages, 2016. doi:10.1155/2016/1487051 PMID: 27651793
2. Chatzopoulou EI,
Roskopf CC, Sekhavati F, Braciak TA, Fenn NC, Hopfner K-P, Fuat S. Oduncu FS,
Fey GH and Rädler JO (2016) Chip-based platform for dynamic analysis of NK cell
cytolysis mediated by a triplebody. Analyst, 141, 2284-2295 PMID: 26958659
3. Oh YJ,
Hubauer-Brenner M, Gruber HJ, Cui Y, Traxler L, Siligan C, Park S and
Hinterdorfer P (2016) Curli mediate bacterial adhesion to fibronectin via
tensile multiple bonds. Sci Rep. 6: 33909 PMID: 27652888
4. Niepel MS, Mano JF
and Groth T (2016) Effect of Polyelectrolyte Multilayers Assembled on Ordered
Nanostructures on Adhesion of Human Fibroblasts. ACS Appl. Mater. Interfaces, 8
(38), pp 25142–25151 PMID: 27603547
5. Ferizi M,
Leonhardt C, Meggle C, Aneja MK, Rudolph C, Plank C, Rädler JO. Stability
analysis of chemically modified mRNA using micropattern-based single-cell
arrays. Lab Chip. 2015 Sep 7;15(17):3561-71. doi: 10.1039/c5lc00749f. Epub 2015
Jul 23. PubMed PMID: 26201602
6. Henderson MJ,
Baldwin HA, Werley CA, Boccardo S, Whitaker LR, Yan X, Holt GT, Schreiter ER,
Looger LL, Cohen AE, Kim DS, Harvey BK. A Low Affinity GCaMP3 Variant (GCaMPer)
for Imaging the Endoplasmic Reticulum Calcium Store. PLoS One. 2015 Oct
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PMID: 26451944; PubMed Central PMCID: PMC4599735
7. Segerer FJ,
Thüroff F, Piera Alberola A, Frey E, Rädler JO. Emergence and Persistence of
Collective Cell Migration on Small Circular Micropatterns. Phys Rev Lett. 2015
Jun 5;114(22):228102. Epub 2015 Jun 2. PubMed PMID: 26196648
8. Pinon P, Pärssinen
J, Vazquez P, Bachmann M, Rahikainen R, Jacquier MC, Azizi L, Määttä JA,
Bastmeyer M, Hytönen VP, Wehrle-Haller B. Talin-bound NPLY motif recruits
integrin-signaling adapters to regulate cell spreading and mechanosensing. J
Cell Biol. 2014 Apr 28;205(2):265-81 PMID: 24778313
9. Röttgermann PJF,
Piera Alberola A and Rädler JO (2014) Cellular self-organization on
micro-structured surfaces. Soft Matter 10, 2397 PMID: 24623049
10. Niepel MS,
Fuhrmann B, Leipner HS, Groth T (2013) Nanoscaled Surface Patterns Influence
Adhesion and Growth of Human Dermal Fibroblasts. Langmuir 13278-13290 PMID: 24090166
11. Sabourin D,
Skafte-Pedersen P, Søe MJ, Hemmingsen M, Alberti M, Coman V, Petersen J, Emnéus
J, Kutter JP, Snakenborg D, Jørgensen F, Clausen C, Holmstrøm K, Dufva M.
(2012) The MainSTREAM Component Platform: A Holistic Approach to Microfluidic
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Cepla V, He C, Edin J, Rakickas T, Kobuch K, Ružele Ž, Jackson WB, Rafat M,
Lohmann CP, Valiokas R, Griffith M. (2015) Functional fabrication of
recombinant human collagen-phosphorylcholine hydrogels for regenerative
medicine applications. Acta Biomaterialia. 12:70-80 PMID: 25448347
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M-P, Cohen AE. Ultrawidefield microscope for high-speed fluorescence imaging
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14. Werley CA, Chien
MP, Gaublomme J, Shekhar K, Butty V, Yi BA, Kralj JM, Bloxham W, Boyer LA,
Regev A, Cohen AE (2017) Geometry-dependent functional changes in iPSC-derived
cardiomyocytes probed by functional imaging and RNA sequencing. PLoS One. 2017
Mar 23;12(3):e0172671. doi: 10.1371/journal.pone.0172671. eCollection 2017. PMID: 28333933
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Zhang H, Werley CA, Cohen AE (2016) Optically Controlled Oscillators in an
Engineered Bioelectric Tissue. Physical Review X, 6, 031001
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FAQ: Handling and Storage of Fibronectin
Navigating the complexities of working with fibronectin can be daunting. Two frequent questions we often encounter are: "Can you freeze fibronectin?" and "How do you dissolve fibronectin properly?" Here's a comprehensive guide to address these queries:
1. How to dissolve fibronectin:
Here's a step-by-step protocol to ensure you get the most out of your fibronectin without causing aggregation:
Begin by adding 5 ml of warm (37°C) water or PBS to the lyophilized fibronectin.
Incubate your sample at 37°C for about 3 hours. Periodically invert the vial during this time. It's crucial to avoid shaking or vortexing, as applying shear force can lead to aggregation.
Once reconstituted, you'll have a solution with 5 mg of soluble fibronectin and a small amount of insoluble fibronectin aggregate. Note: The supplied vial contains slightly over 5 mg to account for the minor quantity that remains undissolved.
Finally, filter your solution slowly through a pre-wetted 0.2 µm membrane filter. Prewetting helps to avoid a premature clogging of a filter
A concentration of 1 mg/ml fibronectin corresponds to an adsorbance A280 of 1.3.
2. Can you freeze fibronectin?
The lyophilised fibronectin should be kept in dry conditions at -20C and is also quite stable at ambient temperature. However once fibronectin is dissolved and sterile filtered it can only be kept at +4C for a month.
Although there's widespread advice suggesting you can freeze fibronectin solution, we strongly advise against it. Fibronectin is a high molecular weight protein, sensitive to freezing. The primary concern is that fibronectin tends to aggregate post-thawing. While it might be tempting to store it in the freezer for extended periods, the potential damage and aggregation could compromise your experiments. Our best practice? Always start with freshly reconstituted and filtered fibronectin. If storage is absolutely necessary, stored at +4°C for a month.
