Name: bead loading proteins and nucleic acids into adherent human cells
Uploaded: 2021-06-01T13:00:00.000+00:00
Duration: 7 min 28 s
Description: Watch this Scientific Journal Video about Bead Loading Proteins and Nucleic Acids into Adherent Human Cells at JoVE.com

We are grateful to the members of the Stasevich lab for innumerable conversations that helped improve and develop this protocol. Specifically, Dr. Linda Forero and Dr. Phil Fox for advice on bead loading different cell types. We would like to sincerely thank Dr. Yoko Hayashi-Takanaka, Dr. Yuko Sato and Dr. Hiroshi Kimura for sharing their glass bead loading protocol. We are very grateful to Dr. Ashok Prasad and Dr. Diego Krapf for generously sharing their bead loading protocols for introducing inorganic particles into cells. We are grateful to Dr. Travis Sanders, Craig Marshall, and Dr. Thomas Santangelo for generously sharing their labeled RNA reagent. ALK, MNS,&#160;CAC, GG&#160;and TJS were supported by the National Institutes of Health (NIH) grant R35GM119728&#160;and the National Science Foundation (NSF) CAREER grant MCB-1845761, both&#160;to TJS. CAC was also supported by the&#160;NSF&#160;NRT award DGE-1450032.

1. Clean, sterilize, and dry glass beads to avoid clumping and ensure even spreading atop the cells.
<ol>
	<li>Sterilize approximately 5 mL of glass beads in sodium hydroxide (NaOH). Measure the beads in a 50 mL conical tube. Add 25 mL of 2 M NaOH and mix gently using a shaker or rotator for 2 h.</li>
	<li>Decant the NaOH, retaining as many beads as possible. If the beads are in suspension, spin down the tube of beads briefly in a centrifuge (1 min at ~1000 &#215; g, room temperature).</li>
	<li>Wash the beads thoroughly with cell culture-grade water until the pH is neutral (use a pH test strip on the eluent to confirm a neutral pH). Decant the wash water each time, as before.</li>
	<li>Wash the beads thoroughly with 100% ethanol 2-3x. Decant the ethanol each time, as before.</li>
	<li>Dry the beads. Sprinkle the beads to form a thin layer inside a sterile container (such as a 10 cm Petri dish). Leaving the container open, let the beads air dry in a biosafety cabinet overnight. Ensure that the beads are completely dry by tapping or gently shaking the container and checking that the beads have a sandy texture with no clumping or flaking.</li>
	<li>UV-sterilize the dry beads for 15 min.</li>
</ol>
2. Assemble the bead loader apparatus.
<ol>
	<li>Fasten a patch of mesh (polypropylene or equivalent material, 105 &#181;m openings to allow the beads to pass through) to cover the entire opening of the beads holding chamber with either tape or clamping the mesh between the male and female ends of a metal reusable imaging chamber (Figure 1A).</li>
	<li>UV-sterilize the apparatus for 15 min. Add the beads to the apparatus and seal it tightly with waxy film. 
	NOTE: It is essential that the beads are completely clean and dry at this step. They should be loose and look sandy with no clumps. If they do not appear so, re-wash and completely dry the beads.</li>
	<li>Store the apparatus in a sealed, dry container desiccated by silica gel or other desiccant medium. If the beads become damp, which will be apparent by bead clumping, thoroughly dry and sterilize the bead loader and replace with fresh beads. 
	&#8203;NOTE: All these precautions will prevent any mold or bacteria from growing on or around the beads within the bead loader. The bead loader apparatus can be made in different ways. See the details in the discussion.</li>
</ol>
3. Prepare glass-bottom chambers of adherent cells.
<ol>
	<li>Seed adherent mammalian cells onto a 35 mm glass-bottom chamber. Ensure that the cells are approximately 80% confluent at the time of bead loading. (See Table 1 for more information on various cell types and notes on the effectiveness of bead loading in different cell types.) 
	NOTE: Cells can be seeded in only the microwell in the center of the chamber to conserve how many cells are used.</li>
	<li>Incubate the cells under normal conditions until they are completely adherent to the glass. 
	&#8203;NOTE: It is essential that the cell density is high enough and that the cells are securely adhered to the glass. If these requirements are not met, cells will likely peel off during bead loading. The timeline between cell seeding and bead loading can be lengthened to ensure proper cell adhesion and confluency.</li>
</ol>
4. Bead loading cells
NOTE: If required, wash the cells briefly with phosphate-buffered saline (PBS) and then add 2 mL of the optimal medium. Incubate for at least 30 min.
<ol>
	<li>Make a solution of 3-8 &#181;L containing the desired plasmids, protein, and/or particles. Use ~1 &#181;g (0.1-1 pmol) of each type of plasmid and ~0.5 &#181;g (0.01 nmol) of protein, depending on experimental requirements. Use a low-retention tube for proteins so that they are not left behind on the tube walls. Bring the solution up to a minimum of 3 &#181;L with PBS, and adjust the solution volume to coat the entire area of cells to be loaded (i.e., the chamber&#39;s microwell, Figure 1B).</li>
	<li>Mix the solution thoroughly by pipetting up and down and/or flicking the tube. Briefly spin the solution down to the bottom of the tube in a tabletop microfuge.</li>
	<li>Transfer the bead loading solution and the chamber of cells into a tissue culture hood. Perform the remaining steps in the tissue culture hood using sterile technique.</li>
	<li>Remove the medium from the cells and temporarily store it in a sterile tube. Gently aspirate all medium from around the edges of the chamber, and tilt the chamber at approximately a 45&#176; angle and remove the remaining drop of media in the center microwell. During medium removal, make sure to avoid letting the pipette tip touch the glass, which may result in cell peeling and loss. Move quickly to the next step so that the cells are not dry for long.</li>
	<li>Gently pipette the bead loading solution onto the glass microwell in the center of the chamber. Optional: Incubate with gentle rocking for ~30 s without allowing the chamber to dry up completely.</li>
	<li>Gently disperse a monolayer of glass beads on top of the cells, preferably using a bead loading apparatus (Figure 1A). Ensure that the beads cover the cells in the glass-bottom microwell completely.</li>
	<li>Pinching the chamber with two fingers, tap it against the hood surface by lifting it ~2 inches and bringing it down firmly. Use a force approximately equivalent to dropping the dish from that height. Repeat for a total of ~10 taps. 
	NOTE: Ensure that the taps do not substantially peel the cells. Tapping can be optimized for the cell type. If cells load poorly, tap harder; however, if many cells peel off, tap more lightly.</li>
	<li>Gently add medium back into the chamber by pipetting slowly onto the plastic side of the chamber. Try to aspirate any floating beads without disturbing the cells. Add more pre-warmed media at this step if too much was removed. Incubate the cells for 0.5-2 h in the incubator.</li>
	<li>UV-sterilize the bead loader for 15 min before returning it to storage under desiccating conditions.</li>
	<li>Add dye (e.g., DAPI or HaloTag ligand stain, if required by the experiment) to the cells as per the manufacturer&#39;s recommended protocol.</li>
	<li>Wash the cells 3x with medium before imaging to remove the beads and excess loading components in solution. Avoid pipetting directly onto cells to keep them from peeling.</li>
</ol>
5. Imaging the bead-loaded cells
<ol>
	<li>Image the cells immediately or when required by the experiment. Use a microscope capable of capturing fluorescence (lasers or monochromatic light source). Ensure that the excitation wavelengths are appropriate for the chosen fluorophore or dye (e.g., 488 nm wavelength light for green fluorescence protein (GFP)). 
	NOTE: Bead-loaded proteins may be imaged once the cells have recovered (as soon as 30 min post loading for the cell lines described here). Plasmid expression takes &#8805;2 h depending on expression vector elements (Figure 1C, and further explanation in the discussion). Imaging of bead-loaded cells can be performed on any microscope equipped with the appropriate fluorescent sources associated with loaded probes, a camera capable of capturing fluorescence images, such as an electron-multiplying charge-coupled device (EMCCD) or scientific complementary metal-oxide semiconductor (sCMOS) camera, and an incubator to control temperature, humidity, and carbon dioxide. For a guide to fluorescence microscopy, refer to 27.</li>
</ol>

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The authors have no conflicts of interest to disclose.

The bead loading technique described here is a cost-effective and time-efficient method for introducing macromolecules and other particles into adherent cells. This versatile process can load protein (Figure 2A)15,16,26,27, a combination of protein and plasmids (Figure 2B,C)22,25, RNA (Figure 4C), 100 and 250 nm polystyrene beads (personal correspondence), synthetic dyes39&#160;or quantum dots34,40. Bead loading may have the capability to load other types of membrane-impermeable particles as well. Its most frequently used application is for loading antibodies or Fabs to target endogenous epitopes, such as post-translational modifications (PTMs), into live cells. Targets, such as PTMs, are often difficult to label in live cells without established PTM-specific, genetically encoded probes41,42. In contrast, bead loading can introduce multiple types of probes, reporters, or other molecular tools together into the same cell for monitoring multiple readouts simultaneously. We anticipate that bead loading will be a useful technique for loading a variety of macromolecules or particles.
A major advantage of bead loading is the low cost: each experiment costs less than 0.01 USD. A bead loader apparatus can be made easily using inexpensive materials costing in total ~$150, which is significantly less expensive than any other cell-loading method. The cost of a bead loader apparatus can be further reduced to under $10 by replacing the reusable metal chamber with a plastic one. For this, either drill a hole in a 35 mm chamber or remove the glass from a 35 mm glass-bottom chamber, then securely fasten the mesh in place with tape. In lieu of an apparatus, bead loading can even be performed using a wide-bore 1000 &#181;L pipette tip to scoop and sprinkle beads onto cells, although this variation makes it difficult to sprinkle a monolayer of beads onto cells (step 4.6).
Another benefit of bead loading is that cells can retain normal overall morphology, recover rapidly, and continue to grow and divide, at least for the U2OS, RPE1, and HeLa cells studied here and for the other cell lines studied elsewhere (Figure 3; Figure 4A,B; Supplemental Video 1; and Table 1)31. During bead loading, cells undergo physical stress and sometimes dislodge and peel (~5% of cells peel under optimal conditions, but greater cell loss can happen if bead loading is performed too forcefully or too many glass beads are loaded atop the cells, as depicted in Figure 3B). However, bead-loaded cells that remain attached to the coverslip usually appear healthy and can be imaged as soon as 30 min after bead loading (Figure 3A). We generally allow cells a 30-min recovery period but anticipate that imaging sooner post-bead loading is feasible.
A major drawback of this technique is that the cells need to be capable of withstanding minor physical stress during loading and remain securely adherent to the coverslip. Poorly/non-adherent cell lines or cells grown on coated plates (e.g., HEK and stem cells) often detach upon gentle tapping during bead loading. Further, experience has shown that primary neurons are too sensitive for bead loading.
Bead loading is best suited for single-cell or single-molecule experiments. In our experience, bead loading has a roughly 20-40% protein loading efficiency, and ~20% of bead-loaded cells also expressed a co-loaded plasmid (Figure 2A,B). Thus, bead loading plasmids may be less efficient for protein expression than bead loading purified proteins because plasmids must not only enter cells but also be expressed (which involves, among other things, nuclear import, transcription, and translation, each of which can lower expression efficiency). The low efficiency of bead-loaded plasmid expression can be circumvented by using alternative transfection protocols, such as lipofection, before bead loading proteins or probes16,27. Additionally, incubating cells in optimal media for 30 min before bead loading may assist plasmid expression. Due to low plasmid expression, bead loading has not often been used as an alternative to lipofection-based transfection in this laboratory. The only exception is when a purified protein, such as Fab, is to be co-loaded, in which case it is quite convenient to bead-load the protein and plasmid at the same time. Moreover, for cells that are unresponsive or intolerant to lipofection, bead loading may provide an alternate, albeit low-efficiency, method for transient plasmid expression.

Loading macromolecules into mammalian cells necessitates methodology that allows them to cross the cell&#39;s plasma membrane1. Several methods can introduce plasmids into mammalian cells through transfection, including liposomal transfection2 and diethylaminoethyl-dextran transfection3. However, methods for loading proteins or membrane-impermeable particles into cells are more limited.
Several techniques have bypassed this difficult hurdle using various strategies. First, microinjection delivers particles through a micropipette into live cells under a microscope4. While arguably the most controlled and least invasive method, this technique is relatively low-throughput because cells must be loaded one by one. Further, microinjection requires specialized equipment and is technically demanding.
Second, electroporation is a way to electro-inject proteins into cells via voltage-induced membrane disruption5,6,7. However, this method again requires specialized, expensive equipment, and the shock can cause cell stress and mortality. Further, cells must be trypsinized before electroporation and subsequently replated, limiting the timeframe at which cells can be investigated post-electroporation.
Third, cell membranes may be chemically modified for temporary, reversible permeabilization8,9. Streptolysin-O loading inserts an endotoxin into cell membranes, which forms temporary pores, allowing exogenous membrane-impermeable particles, including proteins and DNA plasmids, to enter cells10. After a 2 h recovery, about half the cells repair these pores and halt internalizing particles from the solution. However, this technique requires a long recovery time and is incompatible with cell types that cannot tolerate endotoxins.
Fourth, mechanical disruption loads particles into cells through physical perturbation of the cell membrane11. This can be done in multiple ways, including scratching, scraping, and rolling beads atop cells12,13. As early as 1987, beads have been used to load proteins into cells mechanically14. More recently, the bead loading technique has been optimized and adapted beyond proteins to include the loading of plasmids and RNA, as described here.
Bead loading is an easy, inexpensive, and fast method for loading protein and plasmids into adherent human cells. Glass beads are briefly rolled atop cells, temporarily disrupting their cellular membrane. This allows particles in solution to enter. As bead loading has low efficiency, it is best suited for single-molecule or single-cell microscopy experiments. Bead loading can introduce a wide variety of proteins, including fragmented antibodies (Fab),15,16 purified proteins like scFvs,17 intrabodies,18,19, or mRNA coat proteins, e.g., MS2 coat protein (MCP)20,21. Plasmid expression vectors can also be added to the protein solution and bead-loaded simultaneously22,23,24,25.
Beyond proteins and plasmids, molecules as large as 250 nm polystyrene beads have been introduced into cells via bead loading (personal&#160;communication). Bead loading is incredibly inexpensive, costing less than 0.01 USD per experiment in materials and requiring no additional expensive equipment. The cost is further reduced by minimizing the amount of probes used per experiment because only the cells in the central 14 mm-diameter microwell of an imaging chamber are loaded. It should be noted that the limited loading area means that bead loading is not ideal for bulk-cell loading.
This manuscript presents the bead loading process, including how to construct the bead loading apparatus and perform an experiment. It shows that proteins, RNA, and DNA can be loaded into various cell types and that two different, simultaneously bead-loaded proteins have highly correlated cellular concentrations and relatively low variance. Also discussed are variations in the protocol based on cell type and loading of protein, plasmid, or RNA. Although beads are thought to perforate and disrupt the cell membrane, when appropriately performed, the bead loading process dislodges only a small number of cells from the bottom of the imaging chamber. After a short recovery period, cells continue to grow and divide. This methodology is ideal for live-cell microscopy experiments, including single-molecule protein and RNA tracking, post-translational modification detection, observation of dynamic cellular mechanisms, or subcellular localization monitoring15,16,22,26,27.

<table><tbody><tr><td>10 cm cell culture dishes</td><td>VWR</td><td>82050-916</td><td>Use to culture cells</td></tr><tr><td>35 mm cell culture dishes</td><td>Falcon</td><td>353001</td><td>Use to construct bead loader</td></tr><tr><td>Attofluor Cell Chamber</td><td>Thermo Fisher Scientific</td><td>A7816</td><td>Use to construct the custom bead loader</td></tr><tr><td>DMEM, high glucose, no glutamine</td><td>Thermo Fisher Scientific</td><td>11960069</td><td>Use in general cell culture</td></tr><tr><td>Drierite Indicating Absorbents</td><td>Thermo Fisher Scientific</td><td>07-578-3B</td><td>Store the bead loader in a desiccator with these absorbent pellets</td></tr><tr><td>Fetal Bovine Serum</td><td>Atlas Biologics</td><td>F-0050-A</td><td>Use in general cell culture and as a supplement before bead loading</td></tr><tr><td>Glass beads, acid washed, &amp;le;106 &amp;micro;m*</td><td>Millipore Sigma</td><td>G4649</td><td>Sprinkle on cells to bead load plasmid DNA and proteins</td></tr><tr><td>Glass bottom dishes, 35 mm, #1.5, 14 mm glass</td><td>MatTek Corporation</td><td>P35G-1.5-14-C</td><td>Seed cells onto these chambers for imaging</td></tr><tr><td>L-Glutamine-200 mM</td><td>Thermo Fisher Scientific</td><td>25030081</td><td>Use to make DMEM + media</td></tr><tr><td>Opti-MEM, Reduced Serum Medium</td><td>Thermo Fisher Scientific</td><td>31985070</td><td>Optimal media for incubating cells before bead loading (optional step)</td></tr><tr><td>Parafilm</td><td>VWR</td><td>52858-032</td><td>waxy film used to construct bead loader</td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140-122</td><td>Use to make DMEM + media</td></tr><tr><td>Phenol-free DMEM</td><td>Thermo Fisher Scientific</td><td>31053036</td><td>Use on cells before imaging</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Thermo Fisher Scientific</td><td>AM9625</td><td>Working stock of sterile 1X PBS</td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>Thermo Fisher Scientific</td><td>S318-500</td><td>Use 2 M solution to wash glass beads</td></tr><tr><td>Spectra Mesh Woven Filters, Polypropylene, 105 &amp;micro;m opening</td><td>Spectrum Labs</td><td>148496</td><td>Use to construct bead loader</td></tr><tr><td>Trypsin</td><td>Thermo Fisher Scientific</td><td>25300062</td><td>Use in general cell culture</td></tr></tbody></table>

bead loading proteins and nucleic acids into adherent human cells

Many live-cell imaging experiments use exogenous particles (e.g., peptides, antibodies, beads) to label or function within cells. However, introducing proteins into a cell across its membrane is difficult. The limited selection of current methods struggles with low efficiency, requires expensive and technically demanding equipment, or functions within narrow parameters. Here, we describe a relatively simple and cost-effective technique for loading DNA, RNA, and proteins into live human cells. Bead loading induces a temporary mechanical disruption to the cell membrane, allowing macromolecules to enter adherent, live mammalian cells. At less than 0.01 USD per experiment, bead loading is the least expensive cell loading method available. Moreover, bead loading does not substantially stress cells or impact their viability or proliferation. This manuscript describes the steps of the bead loading procedure, adaptations, variations, and technical limitations. This methodology is especially suited for live-cell imaging but provides a practical solution for other applications requiring the introduction of proteins, beads, RNA, or plasmids into living, adherent mammalian cells.

The most common application of bead loading is to introduce one or more types of protein into adherent human cells. To illustrate this, cells were bead-loaded with a solution of a Cy3- and Alexa488-conjugated Fab protein. Although not every cell in the microwell was bead-loaded, the cells that were loaded almost always had both Cy3- and Alexa488-labeled proteins together (Figure 2A). According to an earlier estimate, when 0.5 microgram&#160;of Fab diluted in 4 microliters is bead-loaded29, as in Figure 2A, each cell is loaded with roughly 106 Fab molecules.
Plasmid DNA encoding GFP (1 &#181;g of plasmid DNA, 1.8 &#181;L of a 557 ng/&#181;L solution) and 0.5 &#181;g of Cy3-labeled Fab&#160;was also introduced into cells via bead loading and subsequently expressed and visualized (Figure 2B). The GFP fluorescence indicated that the GFP-encoding plasmid was not only loaded into cells but also expressed. Thus, in the same cell, bead loading can introduce a protein probe (e.g., Cy3-labeled Fab) and reporter plasmid (e.g., GFP), as performed in this laboratory previously22,23,24. We determined that 40% of the cells were bead-loaded with Fab protein and 21% of the bead-loaded cells expressed the co-loaded plasmid, as shown in the representative fields-of-view in Figure 2B. Typically, each chamber is loaded with 1-2 &#181;g of plasmid, approximately the same amount as lipofection.
Bead-loaded cells express widely varying levels of plasmids (Figure 2C,D). To specifically measure this, we used the Fisher Ratio test to compare the distributions of protein and plasmid intensity data. The results showed that although proteins 1 and 2 had similar intensity distributions (p = ~1), each protein had a significantly smaller distribution than the plasmid (p = 3.2e-6 and 1.8e-5). Although this could be due to variability in how many plasmids are loaded per cell, the greater source of variability may arise from the many steps required for plasmid expression that are likely to vary greatly between cells, including being imported into the cell nucleus, transcription, and translation. In contrast, the levels of bead-loaded proteins had slight cell-to-cell variance, and the levels of two simultaneously loaded proteins were highly correlated with each other (Figure 2D,E).
Plasmid expression can be seen as early as 2-4 h post bead loading but may occur later depending on when optimal plasmid expression is obtained. We recommend performing a time course to determine the best window of expression for a specific plasmid spanning 2-24 h post bead loading. This can be done in one chamber with long timeframe imaging or by bead loading and staggering multiple chambers. Bead-loaded cells remain adherent and are healthy enough to grow and divide. Bead-loaded human U2OS cells were imaged directly before, directly after, and 24 h after bead loading. Proper bead loading had almost no noticeable effect on the number of cells or their morphology, as shown in Figure 3A&#160;(left, middle).
In contrast, poor bead loading with too many beads and excessive tapping force is depicted in Figure 3B. This caused much cell loss (large patches of the coverglass without cells and detached, floating, out-of-focus cells), poor cell morphology (cells appearing rounded up and poorly adhered), and clusters of beads remaining on the coverglass after bead loading. Though cells are thought to undergo mechanical damage during bead loading, cells grew and proliferated in the properly bead-loaded chamber, as evidenced by the increased number of cells 24 h after bead loading (Figure 3A, right). The effect on cell viability can be assessed through a variety of assays, such as a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, to compare bead-loaded to mock-loaded cells30. Further, this and previous work show that the bead-loaded cells undergo cell division (Figure 3C and Supplemental Video 1), and the timing of mitosis is not affected by bead loading31, which serves as further evidence for sustained cell health after bead loading.
Bead loading is a versatile technique, accommodating several adherent cell lines and various macromolecules. Here, this variety has been demonstrated by loading RPE1 and HeLa cell lines with Fab (Figure 4A,B). Table 1 provides further examples of bead loading in different cell lines, in this laboratory and beyond, and points out some of the nuanced differences between bead loading protocols from other labs. Of note, the diameter of glass beads used for loading varies greatly between laboratories, though the most efficient loading was found for small, 75 &#181;m diameter beads in several cell lines14. Further, this laboratory has begun bead loading RNA as well (data not shown). Figure 4C displays a representative U2OS cell bead-loaded with a Cy5-RNA 9mer and Cy3-DNA 28mer together.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62559/62559fig01.jpg" /> 
Figure 1: Bead loading apparatus, technique, and timeline <a href="https://www.jove.com/files/ftp_upload/62559/62559fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62559/62559fig02.jpg" /> 
Figure 2. Bead loading introduces low variability in protein concentration but high variability in plasmid expression. (A) Cells were bead-loaded with 0.5 &#181;g of each of Alexa488-conjugated anti-H3K27 acetyl Fab (green) and Cy3-conjugated anti-RNAPII-Serine 5-phosphorylated Fab (red) in 4 &#181;L of bead loading solution. Cells were DAPI-stained (blue) and then live-imaged immediately. Scale bars = 20 &#181;m. (B) Cells were bead-loaded with 0.5 &#181;g of Fab protein (Cy3-conjugated anti-RNAPII-Serine 5-phosphorylated protein, red) and 1 &#181;g of plasmid encoding superfolder GFP-H2B (green) in 4 &#181;L of bead loading solution. After 24 h, cells were DAPI-stained (blue) and imaged live. Scale bars = 30 &#181;m. (C-E) Protein 1 (JF646-HaloLigand-labeled HaloTag-MCP), protein 2 (Cy3-conjugated anti-FLAG Fab), and a plasmid encoding EGFP (&#955;N-EGFP-LacZ) were bead-loaded together into cells. The total intensity in each fluorescent channel was measured in a 1.3 x 1.3 &#181;m patch in the cytoplasm of each cell. N = 25 cells. (C) Representative cells expressing the bead-loaded plasmid, &#955;N-EGFP-LacZ. The same imaging conditions and intensities were used for all cells. Spots are aggregates of the expressed protein. Scale bars = 10 &#181;m. (D) The chart shows each cell&#39;s total intensity of either protein 1, protein 2, or EGFP expressed from the plasmid. Each channel was normalized to the median. Bonferroni-corrected P values were calculated by the Fisher Ratio test to determine whether the distribution of protein or plasmid intensity data has the same variability. Each point represents a cell. (E) The total intensities for either both proteins, protein 1 and the plasmid, or protein 2 and the plasmid, are plotted against each other. Calculated R2 values are displayed. Each point represents a cell. Abbreviations: DAPI = 4&#8242;,6-diamidino-2-phenylindole; EGFP = enhanced green fluorescent protein; A.U. = arbitrary units; MCP = MS2 coat protein; RNAPII = RNA polymerase II. <a href="https://www.jove.com/files/ftp_upload/62559/62559fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62559/62559fig03.jpg" /> 
Figure 3: Bead-loaded cells remain adherent and are healthy enough to grow and divide. (A) U2OS cells were bead-loaded with 0.5 &#181;g of Cy3-conjugated anti-FLAG Fab in 4 &#181;L of bead loading solution. The cells were imaged directly before, directly after bead loading, and 24 h after bead loading. Orange arrows identify areas where cells peeled off during bead loading. Scale bars = 2 mm. (B) Representative image of U2OS cells bead-loaded with components from (A) but with harsh tapping and too many beads. The red arrow identifies extra glass beads. Scale bar = 2 mm. (C) U2OS cells were loaded with 1.5 &#181;g of the 14.4 kbp plasmid smFLAG-KDM5B-15xBoxB-24xMS2, 0.5 &#181;g of Cy3-conjugated anti-FLAG Fab (green), 130 ng of HaloTag-MCP (magenta) in 8 &#181;L of bead loading solution. Directly before imaging, the HaloTag was stained with JF646-HaloLigand. The MS2 stem-loops of the mRNA transcribed from the reporter plasmid are labeled by MCP (magenta spots), and FLAG-tagged translated reporter protein is labeled by anti-FLAG Fab (green colocalization to mRNA). Mature Fab-labeled protein localizes to the nucleus. This cell was imaged 4-15 h after bead loading. Yellow arrows identify the cell nucleus before and nuclei after cell division. Scale bars = 5 &#181;m. Abbreviation: MCP = MS2 coat protein. <a href="https://www.jove.com/files/ftp_upload/62559/62559fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62559/62559fig04.jpg" /> 
Figure 4: Variations in cell type loading material of the bead loading protocol. (A-B) RPE1 (A) and HeLa (B) cells were bead-loaded with 1.5 &#181;g of a nuclear Fab protein (anti-RNAPII-Serine 5-phosphorylation) in 4 &#181;L of loading solution. Each cell&#39;s nucleus (nuc) and cytoplasm (cyto) are marked. Cells were imaged 6 h after being bead-loaded. Scale bars = 5 &#181;m. (C) Human U2OS cells were bead-loaded with both Cy5-RNA 9mer (magenta) and Cy3-DNA 28mer (green) oligos, 10 picomoles of each, in 4 &#181;L of bead loading solution. Cells were imaged 4 h after being bead-loaded. All cell nuclei are highlighted by a dashed line. Scale bars = 5 &#181;m. Abbreviations: RNAPII = RNA polymerase II. <a href="https://www.jove.com/files/ftp_upload/62559/62559fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody style="font-size: 14px;">
		<tr>
			<td>Cell line</td>
			<td>Cell type</td>
			<td>Bead loading effectiveness</td>
			<td>Notes/reference</td>
		</tr>
		<tr>
			<td>Stem cells (human)</td>
			<td>Embryonic stem cells</td>
			<td>Difficult</td>
			<td>*Many cells peel off during bead loading if plated on gelatin-coated plates</td>
		</tr>
		<tr>
			<td>HEK 293</td>
			<td>Human embryonic kidney cells</td>
			<td>Difficult</td>
			<td>*Need to lay down a gel matrix to imaging chamber surface before bead loading. Tap gently when bead loading at first.</td>
		</tr>
		<tr>
			<td>Neurons (rat)</td>
			<td>Primary embryonic neurons (e-18), dissociated</td>
			<td>Very inefficient</td>
			<td>*Efficient bead loading of neurons was not observed using this standard bead loading protocol. This could be due to the non-adherent nature of neurons or from consequent damage to neural processes.</td>
		</tr>
		<tr>
			<td>MDCK (canine)</td>
			<td>Madin-Darby canine kidney cells</td>
			<td>See McNeil and Warder (1987)14</td>
			<td>*Low-efficiency bead loading14</td>
		</tr>
		<tr>
			<td>U2OS (human)</td>
			<td>Osteosarcoma</td>
			<td>Standard bead loading protocol</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa (human)</td>
			<td>Cervical cancer</td>
			<td>Standard bead loading protocol</td>
			<td></td>
		</tr>
		<tr>
			<td>RPE1 (human)</td>
			<td>Epithelial cells immortalized with hTERT</td>
			<td>Standard bead loading protocol</td>
			<td></td>
		</tr>
		<tr>
			<td>HFF (human)</td>
			<td>Primary foreskin fibroblasts</td>
			<td>See Besteiro et al. (2009)31&#160;</td>
			<td>*Modified protocol of tilting instead of tapping31</td>
		</tr>
		<tr>
			<td rowspan="3">BALB/c 3T3, NIH 3T3, and Swiss 3T3 (mouse)</td>
			<td rowspan="3">Embryonic fibroblasts</td>
			<td rowspan="3">See Gilmore and Romer (1996)32, Emerson et al. (2014)33 and McNeil and Warder (1987)14</td>
			<td>*425&#8211;600 &#956;m glass beads reported32</td>
		</tr>
		<tr>
			<td>*Used 200&#8211;300 &#956;m glass beads33</td>
		</tr>
		<tr>
			<td>*75 &#956;m glass beads gave better results than 400 &#956;m14</td>
		</tr>
		<tr>
			<td>DM (Indian muntjac)</td>
			<td>Skin fibroblasts</td>
			<td>See Manders, Kimura, and Cook (1999)34</td>
			<td></td>
		</tr>
		<tr>
			<td>CHO (hamster)</td>
			<td>Epithelial-like ovary cells</td>
			<td>See Memedula and Belmont (2003)35</td>
			<td>*Used 425&#8211;600 &#956;m glass beads35</td>
		</tr>
		<tr>
			<td>BAE (bovine)</td>
			<td>Bovine aortic endothelial cells (BAEC-11)</td>
			<td>See McNeil and Warder (1987)14</td>
			<td>*75 &#956;m glass beads gave better results than 400 &#956;m14</td>
		</tr>
		<tr>
			<td>PtK-2 (Potomus tridaclylis)</td>
			<td>Epithelial kidney cells</td>
			<td>See McNeil and Warder (1987)14</td>
			<td>*75 &#956;m glass beads gave better results than 400 &#956;m14</td>
		</tr>
		<tr>
			<td>HUVEC (human)</td>
			<td>Umbilical vein endothelial cells</td>
			<td>See Gilmore and Romer (1996)32</td>
			<td>*Used 425&#8211;600 &#956;m glass beads32</td>
		</tr>
		<tr>
			<td>J774 and J774.2 (mouse)</td>
			<td>monocyte macrophage cells</td>
			<td>See Becker et al. (2005)36 and McNeil and Warder (1987)14</td>
			<td>*Gentle agitation (instead of tapping) and 425&#8211;600 &#956;m glass beads36</td>
		</tr>
		<tr>
			<td>MS-5 (mouse)</td>
			<td>bone marrow stromal cells</td>
			<td>See Molenaar et al. (2003)37</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">WPE1-NB11 (human)</td>
			<td rowspan="2">prostate epithelial cells</td>
			<td>See Gilmore and Romer (1996)32 and</td>
			<td>*Used 425&#8211;600 &#956;m glass beads32</td>
		</tr>
		<tr>
			<td>Emerson et al. (2014)33</td>
			<td>*Used 200&#8211;300 &#956;m glass beads33</td>
		</tr>
	</tbody>
</table>
Table 1: Bead loading in different cell lines. For the cell lines that have not yet been bead-loaded in this laboratory, references and notes on variations in the protocol are provided.
Supplemental Video 1: Example of a bead-loaded cell undergoing cell division. U2OS cells were loaded with 1.5 &#181;g of the 14.4 kbp plasmid smFLAG-KDM5B-15xBoxB-24xMS2, 0.5 &#181;g of Cy3-conjugated anti-FLAG Fab (green), 130 ng of HaloTag-MCP (magenta) in 8 &#181;L of bead loading solution. Directly before imaging, the HaloTag was stained with JF646-HaloLigand. The MS2 stem-loops of the mRNA transcribed from the reporter plasmid are labeled by MCP (magenta spots), and FLAG-tagged translated reporter protein is labeled via anti-FLAG Fab (green colocalization to mRNA). Mature Fab-labeled protein localizes to the nucleus. This cell was imaged 4-15 h after bead loading. Scale bar = 10 &#181;m. Abbreviation: MCP = MS2 coat protein. <a href="https://www.jove.com/files/ftp_upload/62559/bead_load_cell_divides.avi" target="_blank">Please click here to download this Video.</a>

Watch this Scientific Journal Video about Bead Loading Proteins and Nucleic Acids into Adherent Human Cells at JoVE.com

Bead Loading Proteins and Nucleic Acids into Adherent Human Cells

Bead loading introduces proteins, plasmids, and particles into adherent mammalian cells. This cell loading technique is inexpensive, rapid, and does not substantially affect cell health. It is best suited for live-cell imaging.

Many live-cell imaging experiments use exogenous particles (e.g., peptides, antibodies, beads) to label or function within cells. However, introducing ...

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4.7K Views.  Colorado State University. Bead loading introduces proteins, plasmids, and particles into adherent mammalian cells. This cell loading technique is inexpensive, rapid, and does not substantially affect cell health. It is best suited for live-cell imaging. 

Video: Bead Loading Proteins and Nucleic Acids into Adherent Human Cells

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Bioengineering

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Immunology and Infection

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) soluble cytokines. T cells release two kinds of effector particles in response to activation: trans-synaptic vesicles (tSVs) and supramolecular attack particles, which transfer intercellular messengers and mediate cytotoxicity, respectively. These entities are quickly internalized by APCs engaged in physical contact with T cells, making their characterization daunting. This paper presents the protocol to fabricate and use Bead-Supported Lipid Bilayers (BSLBs) as antigen-presenting cell (APC) mimetics to capture and analyze these trans-synaptic particles. Also described are the protocols for the absolute measurements of protein densities on cell surfaces, the reconstitution of BSLBs with such physiological levels, and the flow cytometry procedure for tracking synaptic particle release by T cells. This protocol can be adapted to study the effects of individual proteins, complex ligand mixtures, pathogen virulence determinants, and drugs on the effector output of T cells, including helper T cells, cytotoxic T lymphocytes, regulatory T cells, and chimeric antigen receptor-expressing T cells (CART).

Here we present the protocol for the stepwise reconstitution of synthetic antigen-presenting cells using Bead-Supported Lipid Bilayers and their use to interrogate the synaptic output from activated T cells.

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) ...

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of ...

Bead Loading to Introduce Nucleic Acids Into Adherent Cultured Cells: A Technique to Load Fluorescent Protein-Encoding Plasmids Into Adherent Mammalian Cells

Source: Cialek, C. A. et al. <a href="https://www.jove.com/v/62559">Bead Loading Proteins and Nucleic Acids into Adherent Human Cells.</a> J. Vis. Exp. (2021).
In this video, we describe the procedure to introduce nucleic acids into adherent mammalian cells using bead loading.

Source: Cialek, C. A. et al. Bead Loading Proteins and Nucleic Acids into Adherent Human Cells. J. Vis. Exp. (2021).
In this video, we describe the ...

Bead Loading Proteins and Nucleic Acids into Adherent Human Cells

Summary

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Chapters in this video

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Biomimetic Materials to Characterize Bacteria-host Interactions

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Bead Loading to Introduce Nucleic Acids Into Adherent Cultured Cells: A Technique to Load Fluorescent Protein-Encoding Plasmids Into Adherent Mammalian Cells