This work is supported by the BBSRC, EPSRC, MRC and Wellcome Trust. The C.B. laboratory is part of the Wellcome Trust Centre for Cell-Matrix research, University of Manchester, which is supported by core funding from the Wellcome Trust (grant number 088785/Z/09/Z). The authors wish to acknowledge the funding provided by the Biotechnology and Biological Sciences Research Council (BBSRC) to C.M., K.J. (BB/M020630/1) and P.A. (BB/P000681/1) and by the Engineering and Physical Sciences Research Council (EPSRC) and Medical Research Council (MRC) Centre for Doctoral Training in Regenerative Medicine to A.K. (EP/L014904/1). The authors thank Alv&#233;ole for their correspondence and their after-sales support team. The authors thank Peter March and Roger Meadows from the Bioimaging Facility, University of Manchester for their help with the microscopy. The Bioimaging Facility microscopes used in this study were purchased with grants from BBSRC, Wellcome Trust and the University of Manchester Strategic Fund.

The authors have nothing to disclose.

LIMAP (PRIMO) micro-patterning advantages and comparison with microcontact printing 
While microcontact printing is possibly the most commonly used micro-patterning technique in the biological field39, there seems to be an increasing number of researchers using LIMAP technology40,41,42,43,44. Here, we presented a protocol using PRIMO, a commercially available system for LIMAP. Below we briefly discuss potential advantages and limitations of microcontact printing and LIMAP photo-patterning.
Microcontact printing requires lithographed masters produced by spin-coating a photo-mask (generally SU-8) onto a glass or silicon wafer, which is then laser-etched with the desired micro-features. These masters are used as templates to create a PDMS stamp45. The stamp is incubated with a chosen protein that adsorbs to it, and is then transferred (stamped) onto the cell culture dish. The process of adsorption of the protein to the PDMS stamp is dependent on protein concentration, buffer and incubation time. These parameters need to be tested beforehand for optimal results46.
Masters can be used in a substantial number of experiments, lasting for months or even years, if correctly preserved. However, a limiting factor of this technology is the necessity to re-design new lithographed masters for every desired modification. Changes in experimental designs may result in time-consuming production of new masters (up to several weeks) thus delaying experiments. In comparison, LIMAP photopatterning does not require a physical master; it uses software-generated pattern templates that can be used to flexibly adapt desired geometries of micro-patterns to changing research questions. LIMAP can be also used to generate protein gradients within the same micro-pattern (Figure 8), which is harder to obtain in a reproducible manner using microcontact printing47.
Furthermore, the micro-pattern resolution achieved with LIMAP, in our case, is 2 &#181;m (Figure 6B).
Approaching this resolution increased intra- and inter- pattern variability. Generating patterns around or above 10 &#181;m width was highly reproducible (Figure 6G,H). On the contrary, with microcontact printing it is difficult to consistently obtain resolutions below 10 &#181;m and it is common to find artefacts when stamping small features (data not shown).
We have shown that LIMAP can be used to micro-pattern multiple proteins (Figure 9) within the same micro-well, allowing further levels of complexity to be added to experiments. Although this could be achieved with microcontact printing, aligning different proteins with high level of precision can be technically rather demanding. Whilst patterning multiple proteins using LIMAP seems straight forward, it is important to mention that cross-binding of proteins through sequential coating procedures can be reduced through blocking reagents but not entirely eliminated (Figure 9).
Regarding the cost of one or the other technique, LIMAP as described here requires the purchase of micro-patterning equipment (PRIMO) that can be installed on different fluorescence microscopes and requires a motorized stage. Although this investment is initially cost intensive, there are no additional purchases other than consumable items (stencils, PEG and PLPP) on the long run associated with LIMAP. Alternatively, the PDMS stencils can also be produced in the lab by the own experimenter following published protocols18,32. The largest costs for microcontact printing may be associated with the production of new masters, which can become substantial if experiments require new patterns.
One drawback of LIMAP is the relatively low throughput approach of this technique. Microcontact printing can produce a large number of micro-patterns quickly and efficiently in a simultaneous stamping step, compared to the required sequential laser micro-patterning with LIMAP. For example, it is possible to produce 6 stamped glass coverslips in about 2 h with microcontact printing using PDMS stamps (excluding stamp preparation); patterning a similar area (6-well dish) with LIMAP would take around 4 h, excluding the procedure of surface passivation (considering the pattern template configuration described in step 5.12 and see Figure 5B).
Another rate limiting factor of LIMAP technology is the long illumination time required for patterning large areas (30 s per design unit with a 7.5 mW/mm2 laser). In these cases, microcontact printing might be a preferred option. A newly available photo-initiator (PLPP gel, Table of Materials) should considerably reduce the time taken for the patterning, allowing the generation of hundreds of micro-patterns in large areas (up to 8 mm2) in just a few minutes.
Another important factor to take into account when micro-patterning surfaces for cell culture is the reproducibility of the micro-patterns among different experimental repeats, in comparison to the variability obtained with microcontact printing. For example, the graphs shown in Figure 7B,D are representative data of three independent experimental repeats with very similar results (data not shown). Based on our experience and previous publications, this level of reproducibility is difficult to achieve with microcontact printing48,49,50,51,52.
In contrast to other photo-patterning techniques that require either dedicated chemistry to engineer photosensitive materials or the use of photo-sensitizers, which are generally not very biocompatible3, the photosensitive component of LIMAP (PLPP) is biocompatible and well-tolerated by cells21; in our hands we have not experienced any cytotoxicity across a variety of cells, including CAD, DRG neurons (Figure 10), fibroblasts, epithelial cells, and melanoma cells (data not shown). Another advantage of LIMAP using PRIMO compared to other photo-patterning techniques is that no photomask is required. Similar to microcontact printing, new photomasks would need to be designed and generated for every desired pattern.
All the limitations mentioned above for microcontact printing, refer to the manual approach of the technique. However, it is possible to enhance the throughput and the reproducibility of microcontact printing using an automated device with stamp load and pressure control53.
Key steps of the protocol and problems solving for LIMAP using PRIMO 
One of the most common problems found during this protocol is having high levels of background fluorescence within the micro-patterns. This can be due to the drying out of micro-wells which often occurs due to their small volume. When this occurs, PBS crystals often appear surrounding the ECM patterns (Figure 11A).
Insufficient or inefficient washing steps after protein incubation can also result in high levels of background fluorescence. This can be observed particularly upon using protein concentrations of 10 &#181;g/mL (Figure 11B) or higher. The excess of protein in the background can be reduced by including additional washing steps with PBS.
The presence of protein background needs to be measured and characterized in each experiment, calculating the background fluorescence intensity (Figure 6E) and subtracting it from the micro-patterns intensity (Figure 6F-H and Figure 7B,D). High protein background may have an impact in the attachment and sprouting of CAD cells, compromising the interpretation of results.
Having gaps between design units is a common problem when users have limited experience (Figure 11B), which occurs as a result of insufficient overlap between patterns. Two parameters in the Leonardo software can be adjusted to overcome this: 1) a negative spacing between columns may be required, depending on the design of the pattern (step 5.7 and see Figure 5B,C). Alternatively, 2) use the gradient option in the Expert menu to stitch the columns. A quick test to determine the optimal spacing parameters can be performed using UV adhesive (Table of Materials). A small drop of this adhesive is applied to a glass slide, which is then covered with a glass coverslip, making a film. The embedded UV adhesive is photopatterned with the pattern template of interest using a low laser dose (30 mJ/mm2). The UV- exposed regions of the embedded adhesive will be cured, becoming visible under bright-field microscopy. The test results are visualized to evaluate the obtained spacing within the pattern. In our neuronal experiments, a gap between stripes may adversely affect cell behavior, producing variations in growth dynamics (either reduced speed or abandonment of the path).
In the latest update of Leonardo software (at the time of publication, Leonardo 4.11), it is possible to upload previously designed bigger pattern templates that cover a much larger area (up to 8 mm2 using the 20X objective) of the micro-well surface compared to the current 0.1 mm2 per design unit, eliminating the need to stitch together the smaller design units. Undefined edges can result from a lack of laser focus adjustment during pattern generation (Figure 11C). It is therefore critical to calibrate the laser and perform reference pattern steps (see step 4) prior to patterning. Poorly defined stripes result in variations in stripe width, making the correlation between axon growth dynamics and stripe width difficult. Axons also tend to abandon stripes that have diffused edges. Additionally, variability in edges can also be found when printing stripes of 10-20 &#181;m width or higher, resulting in a higher protein content at the edges compared to the central regions of the pattern (Figure 6B,D). This edge effect is produced by a non-homogeneous diffusion of the photo-initiator during the photopatterning process. The photoscission reaction is oxygen dependent, which diffuses more at the edges. This edge effect can be minimized homogenizing the photo-initiator with a pipette in the micro-well during the photopatterning process. Furthermore, a new commercialized photo-initiator (PLPP gel), can also reduce the edge effect (PRIMO system support team, personal communication).
Micro-printing of more than one protein can result in cross-binding (Figure 9A-D). This can be minimized by increasing the blocking efficiency that is used to occupy unspecific binding sites between the incubation steps for the two different proteins. Cross-binding of proteins can perturb reproducibility of experimental outcomes and may lead to misinterpretation of data, since it is difficult to determine the contribution of each protein to axon growth dynamics and to other cell behaviors.
Conclusion 
We hope that the provided protocol using LIMAP facilitates the generation of protein micro-patterns through the use of the PRIMO system. Whilst our protocol focuses on how to reliably produce micro-patterns in 2D glass surfaces, others have shown that it is possible to use LIMAP for micro-patterning of soft substrates54, and microstructured surfaces for 3D cultures42. These micro-patterns can be a versatile tool to study cellular responses to changes in their micro-environment.

In recent years, the biological sciences have increasingly made use of the advances provided by material sciences. One prominent example is the micro-patterning of substrates, which can be used to study cellular responses such as cell proliferation1,2, differentiation3,4,5,6, cell migration7,8,9 and pathfinding10,11. There are a number of techniques available that enable the micro-patterning of substrates, such as multiphoton excited photochemistry12, AFM dip-pen nanolithography13, pin and inkjet direct printing14, electron beam lithography15 or microfluidics16. However, two techniques that are widely used in the biological field are microcontact printing17,18,19 or laser-assisted patterning3 (Figure 1). Laser-assisted patterning is considered to deliver more reliable results in terms of protein and PEG stability and cell confinement on the patterns, compared to microcontact printing20. A more novel approach for micro-patterning described here is the use of Light-Induced Molecular Adsorption of Proteins21 (LIMAP, Figure 1D) using a commercially available system (PRIMO, Table of Materials). Each of the methods has advantages and limitations that are briefly described below.Microcontact printing uses PDMS molds (stamps) with desired micro-features that are generated from lithographed masters. The stamps are incubated with a chosen protein which is then transferred (stamped) onto the cell culture substrate18 (Figure 1A). Laser-assisted patterning uses UV light to cleave an anti-fouling film22,23,24,25, exposing regions that can subsequently be coated with the protein of interest (Figure 1B). While the resolution achieved with photo-patterning approaches is in the micron range25,26, most of these techniques require a photo-mask, either in contact with the sample, or situated in the object plane of the microscope objective23,27,28. The requirements for masks in both microcontact printing and photo-patterning can be a limitation; specific masks are required for every geometric pattern and size, which can be expensive and time consuming to generate. In contrast to these techniques, LIMAP does not require a mask (Figure 1D). Using the PRIMO system for LIMAP can be cost intensive at the beginning because it requires purchasing of equipment. However, open-source software is used to design patterns of any desired geometry, giving much more freedom and allowing more complex experiments including the use of protein concentration gradients.The PRIMO laser is controlled and directed by a digitally-controlled micromirror device (DMD) to create patterns in any number of user-defined geometries. LIMAP requires the culture surface to be coated with molecules that prevent cell attachment. Polyethylene glycol (PEG) is most commonly used as such an &#34;antifouling&#34; reagent; it forms a dense anti-adhesive film on the glass or plastic surface29. Subsequently, a photo-initiator is added that allows the PEG film to be removed with high precision through a photoscission mechanism30 by local exposure to UV light under the control of the DMD. These PEG-free regions can be coated with proteins that adsorb to the laser-etched surface, generating a micro-pattern. By varying the laser power, different quantities of PEG can be removed from the surface allowing the user to generate protein gradients. PEG removal and the coating procedure can be repeated to create patterns with two or more distinct proteins in the same micro-well21.The generated micro-patterns provide adhesive surfaces for cells, allowing the study of cell behavior. In our studies, we use micro-patterning to study neurite or axon pathfinding of a neuronal cell line (CAD (Cathecholaminergic-a differentiated) cells31) or primary rat dorsal-root ganglion (DRG) neurons, respectively. Here, we outline a step-by-step protocol for LIMAP (Figure 2) using the commercially available PRIMO system and accompanying Leonardo software. We demonstrate how it can be used for the generation of patterns with defined geometries and multiple proteins, which we use to study axonal pathfinding. We discuss common issues that can arise and how these can be avoided.

<table><tbody><tr><td>Alexa 488 protein labeling kit</td><td>Invitrogen</td><td>A10235</td><td>Working concentration: N.A.</td></tr><tr><td>Alexa 647 protein labeling kit</td><td>Invitrogen</td><td>A20173</td><td>Working concentration: N.A.</td></tr><tr><td>CAD cells</td><td>ECACC</td><td>8100805</td><td>Working concentration: N.A.</td></tr><tr><td>Conjugated fibrinogen-488</td><td>Molecular Probes</td><td>F13191</td><td>Working concentration: 10 &amp;mu;g/ml</td></tr><tr><td>DMEM culture medium</td><td>Gibco</td><td>11320033</td><td>Working concentration: N.A.</td></tr><tr><td>Epifluorescence Microscope**</td><td>Nikon</td><td>Eclipse Ti inverted</td><td>Working concentration: N.A.</td></tr><tr><td>Fibronectin</td><td>Sigma</td><td>F4759</td><td>Working concentration: 10 &amp;mu;g/ml&lt;br/&gt; (after labelling with Alexa 488 protein labeling kit, see above)&lt;br/&gt; (diluted in PBS)</td></tr><tr><td>Fiji-Image J</td><td>&lt;a href=&quot;http://www.imagej.nih.gov&quot; target=&quot;_blank&quot;&gt;www.imagej.nih.gov&lt;/a&gt;</td><td>Version 2.0.0-rc-54/1.51f</td><td>Working concentration: N.A.</td></tr><tr><td>Fluorescent highlighter</td><td>Stabilo</td><td>Stabilo Boss Original</td><td>Working concentration: N.A.</td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630080</td><td>Working concentration: 1M</td></tr><tr><td>Inkscape software</td><td>Inkscape</td><td>Check last update</td><td>Working concentration: N.A.</td></tr><tr><td>Laminin-red fluorescent rhodamine</td><td>Cytoskeleton, Inc.</td><td>LMN01</td><td>Working concentration: 10 &amp;mu;g/ml&lt;br/&gt; (diluted in PBS)</td></tr><tr><td>Leonardo software</td><td>Alv&amp;eacute;ole</td><td>version 4.11</td><td>Working concentration: N.A.</td></tr><tr><td>L-Glutamine</td><td>Sigma</td><td>G7513</td><td>Working concentration: 1%</td></tr><tr><td>Micro-manager software</td><td>Open imaging</td><td>Check last update</td><td>Working concentration: N.A.</td></tr><tr><td>Motorized x/y stage</td><td>PRIOR Scientific</td><td>Proscan II</td><td>Working concentration: N.A.</td></tr><tr><td>NIS Elements Software</td><td>Nikon</td><td>NIS Elements AR 4.60.00 64-bit (With Nikon jobs)</td><td>Working concentration: N.A.</td></tr><tr><td>PBS (without Ca&lt;sup&gt;2+&lt;/sup&gt;, Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td>Sigma</td><td>D8537</td><td>Working concentration: 1X</td></tr><tr><td>PDMS Stencils</td><td>Alv&amp;eacute;ole</td><td>visit &lt;a href=&quot;http://www.alveolelab.com&quot; target=&quot;_blank&quot;&gt;www.alveolelab.com&lt;/a&gt;</td><td>Working concentration: N.A.</td></tr><tr><td>PEG-SVA</td><td>Laysan bio, Inc.</td><td>MPEG-SVA-5000-1g</td><td>Working concentration: 50 mg/ml</td></tr><tr><td>Phalloidin 405</td><td>Abcam</td><td>ab176752</td><td>Working concentration: 1:1000</td></tr><tr><td>Photo-initiator (PLPP)</td><td>Alv&amp;eacute;ole</td><td>Classic PLPP</td><td>Working concentration: 14.5 mg/ml</td></tr><tr><td>Photo-initiator (PLPP gel)</td><td>Alv&amp;eacute;ole</td><td>PLPP gel</td><td>Working concentration: 4.76% diluted in ethanol</td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC-32G (115V) | PDC-32G-2 (230V)</td><td>Working concentration: N.A.</td></tr><tr><td>PLL-PEG</td><td>SuSoS (also distributed by Alv&amp;eacute;ole)</td><td>&lt;a href=&quot;http://www.alveolelab.com&quot; target=&quot;_blank&quot;&gt;www.alveolelab.com&lt;/a&gt;</td><td>Working concentration: 0.1 mg/ml (diluted in PBS)</td></tr><tr><td>Poly-L-Lysine</td><td>Sigma</td><td>&lt;a href=&quot;https://www.sigmaaldrich.com/ProductLookup.html?ProdNo=P4707&amp;amp;Brand=SIGMA&quot;&gt;P4707&lt;/a&gt;</td><td>Working concentration: 0.01%</td></tr><tr><td>Primo equipment</td><td>Alv&amp;eacute;ole</td><td>&lt;a href=&quot;http://www.alveolelab.com&quot; target=&quot;_blank&quot;&gt;www.alveolelab.com&lt;/a&gt;</td><td>Working concentration: N.A.</td></tr><tr><td>Pen/Strep</td><td>Thermo Fisher</td><td>&lt;a href=&quot;https://www.thermofisher.com/order/catalog/product/15140122&quot;&gt;15140122&lt;/a&gt;</td><td>Working concentration: 1%</td></tr><tr><td>Tubulin anti-alpha antibody</td><td>Abcam</td><td>DM1A</td><td>Working concentration: 1:1000&lt;br/&gt; CAD cells</td></tr><tr><td>Tubulin anti-beta 3 antibody</td><td>Sigma</td><td>T8660</td><td>Working concentration: 1:500&lt;br/&gt; DRG neurons</td></tr><tr><td>UV adhesive</td><td>Norland Products</td><td>NOA81</td><td>Working concentration: N.A.</td></tr><tr><td>1 well glass bottom dish</td><td>Cellvis</td><td>D35-20-1.5-N</td><td>Working concentration: N.A.</td></tr><tr><td>6 well glass bottom dish</td><td>Cellvis</td><td>P06-20-1.5-N</td><td>Working concentration: N.A.</td></tr><tr><td>20x objective**</td><td>Nikon</td><td>no phase ring (check updated catalogue)</td><td>Working concentration: N.A.&lt;br/&gt; **Epifluorescence microscope: images were acquired and patterns were generated on an Eclipse Ti inverted microscope (Nikon), coupled to PRIMO micro-patterning equipment (Alv&amp;eacute;ole), using a 20x objective (0.75 S Plan Fluor (nophasering, Nikon). Nikon specific filter sets for GFP, mCherry and Cy5 were used and fluorescent light source was LED (Lumencor) although other fluorescence sources and filter sets can be used. The microscope has an automated x/y stage (PRIOR Scientific) for the printing of multi-field patterning and Nikon Perfect Focus to prevent focus drift. The images were collected using a Retiga R6 (Q-Imaging) camera.</td></tr></tbody></table>

light-induced molecular adsorption of proteins using the primo system for micro-patterning to study cell responses to extracellular matrix proteins

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the cells themselves. Here, we present the method of Light-Induced Molecular Adsorption of Proteins (LIMAP) using the PRIMO system as a patterning technique to produce micro-patterned extracellular matrix (ECM) substrates using a single or combination of proteins. The method enables printing of ECM patterns in micron resolution with excellent reproducibility. We provide a step-by-step protocol and demonstrate how this can be applied to study the processes of neuronal pathfinding. LIMAP has significant advantages over existing micro-printing methods in terms of the ease of patterning more than one component and the ability to generate a pattern with any geometry or gradient. The protocol can easily be adapted to study the contribution of almost any chemical component towards cell fate and cell behavior. Finally, we discuss common issues that can arise and how these can be avoided.

Watch this Scientific Journal Video about Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins at JoVE.co

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

Our overall aim is to understand how cells sense extracellular cues that lead to directed axonal growth. Here, we describe the methodology of Light-Induced Molecular Adsorption of Proteins, used to produce defined micro-patterns of extracellular matrix components in order to study specific events that govern axon outgrowth and pathfinding.

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the ...

light-induced-molecular-adsorption-proteins-using-primo-system-for

Research

JoVE Journal

Neuroscience

12.0K Views.  The University of Manchester. Micro-patroon is een zeer krachtig instrument, omdat het mogelijk maakt het bestuderen van cellulaire reacties om biochemische en biofysische signalen die cellen tegenkomen in levende organismen te definiëren. In tegenstelling tot andere methoden is de beschreven flexibeler voor patroongeneratie. Het maakt bijvoorbeeld de productie van gradiënten en micropatronen van meerdere eiwitten met een hoge reproduceerbaarheid mogelijk.Onze groep gebruikt LIMAP niet alleen om neuronale pathfin...

Video: Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

Biochemistry

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.

A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination.

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of ...

Bioengineering

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe a novel protocol employing the TiO2 photocatalysis to locally alter cell affinity of the substrate surface. For this experiment, a thin TiO2 film was sputter-coated on a glass coverslip, and the TiO2 surface was subsequently modified with an organosilane monolayer derived from octadecyltrichlorosilane (OTS), which inhibits cell adhesion. The sample was immersed in a cell culture medium, and focused UV light was irradiated to an octagonal region. When a neuronal cell line PC12 cells were plated on the sample, cells adhered only on the UV-irradiated area. We further show that this surface modification can also be performed in situ, i.e., even when cells are growing on the substrate. Proper modification of the surface required an extracellular matrix protein collagen to be present in the medium at the time of UV irradiation. The technique presented here can potentially be employed in patterning multiple cell types for constructing coculture systems or to arbitrarily manipulate cells under culture.

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Biology

Expanding the Mass Photometry Analysis Concentration Range with Microfluidics

Analysis of Protein Complex Formation at Micromolar Concentrations by Coupling Microfluidics with Mass Photometry

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without labels. Mass photometry is generally suited to analyzing samples in the 100 pM-100 nM concentration range. However, in many biological systems, it is necessary to measure more concentrated samples to study low-affinity or transient interactions. Here, we demonstrate a method that effectively expands the range of sample concentrations that can be analyzed by mass photometry from nanomolar to tens of micromolar. 
In this protocol, mass photometry is combined with a novel microfluidics system to investigate the formation of protein complexes in solution in the micromolar concentration range. With the microfluidics system, users can maintain a sample at a desired higher concentration followed by dilution to the nanomolar range - several milliseconds prior to the mass photometry measurement. Due to the speed of the dilution, data is obtained before the equilibrium of the sample has shifted (i.e., dissociation of the complex). 
The technique is applied to measure interactions between an immunoglobulin G (IgG) antibody and the neonatal Fc receptor, showing the formation of high-order complexes that were not quantifiable with static mass photometry measurements. 
In conclusion, the combination of mass photometry and microfluidics makes it possible to characterize samples in the micromolar concentration range and is proficient in measuring biomolecular interactions with weaker affinities. These capabilities can be applied in a range of contexts - including the development and design of biotherapeutics - enabling thorough characterization of diverse protein-protein interactions.

Author Spotlight: Characterization of Low-Affinity Protein Interactions in Solution Using MassFluidix Technology

This protocol combines mass photometry with a novel microfluidics system to investigate low-affinity protein-protein interactions. This approach is based on the rapid dilution of highly concentrated complexes in solution, which enables low-affinity measurements and broadens the applicability of mass photometry.

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand ...

Light-Controlled Reversible Protein Patterning on Lipid Membranes

Dynamic Light-Induced Protein Patterns at Model Membranes

The precise localization and activation of proteins at the cell membrane at a certain time gives rise to many cellular processes, including cell polarization, migration, and division. Thus, methods to recruit proteins to model membranes with subcellular resolution and high temporal control are essential when reproducing and controlling such processes in synthetic cells. Here, a method is described for fabricating light-regulated reversible protein patterns at lipid membranes with high spatiotemporal precision. For this purpose, we immobilize the photoswitchable protein iLID (improved light-inducible dimer) on supported lipid bilayers (SLBs) and on the outer membrane of giant unilamellar vesicles (GUVs). Upon local blue light illumination, iLID binds to its partner Nano (wild-type SspB) and allows the recruitment of any protein of interest (POI) fused to Nano from the solution to the illuminated area on the membrane. This binding is reversible in the dark, which provides dynamic binding and release of the POI. Overall, this is a flexible and versatile method for regulating the localization of proteins with high precision in space and time using blue light.

Author Spotlight: Photo Switchable Protein Recruitment for Reversible Patterning in Artificial Cellular Systems

Here, a protocol is described for generating light-regulated and reversible protein patterns with high spatiotemporal precision at artificial lipid membranes. The method consists of the localized photoactivation of the protein iLID (improved light-inducible dimer) immobilized on model membranes that, under blue light, binds to its partner protein Nano (wild-type SspB).

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

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