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 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.
The rolling adhesion cascade describes how circulating cells tether to and roll along the blood vessel wall1. Passive rolling is primarily mediated by selectins, a major class of cellular adhesion molecules (CAMs)1. Under the shear flow of blood, leukocytes expressing P-selectin glycoprotein ligand-1 (PSGL-1) form highly transient bonds with P-selectin, which may be expressed on the surface of inflamed endothelial cells. This process is critical for leukocytes to migrate to a site of inflammation2. In addition, PSGL-1 is also a mechanosensitive receptor capable of triggering the subsequent firm adhesion stage of the rolling adhesion cascade upon its engagement with P-selectin3.
Genetic mutations affecting CAM function can severely affect the immune system, such as in the rare disease of leukocyte adhesion deficiency (LAD), where malfunction of adhesion molecules mediating rolling leads to severely immunocompromised individuals4,5,6. In addition, circulating tumor cells have been shown to migrate following a similar rolling process, leading to metastasis7,8. However, because cell rolling is fast and dynamic, conventional experimental mechanobiology methods are unsuitable for studying molecular interactions during cell rolling. While single-cell and single-molecule manipulation methods like atomic force microscopy and optical tweezer were able to study molecular interactions such as P-selectin's force-dependent interaction with PSGL-1 at the single-molecule level9, they are unsuitable for investigating live adhesion events during cell rolling. Additionally, the interaction characterized in vitro cannot directly answer the question about molecular adhesion in vivo. For instance, what molecular tension range is biologically relevant when cells are functioning in their native environment? Computational methods such as adhesive dynamics simulation10 or simple steady-state model11 have captured certain molecular details and how they influence the rolling behavior but are highly dependent on the accuracy of the modeling parameters and assumptions. Other techniques such as traction force microscopy can detect forces during cell migration but do not provide sufficient spatial resolution or quantitative information on molecular tension. None of these techniques can provide direct experimental observations of the temporal dynamics, spatial distribution, and magnitude heterogeneity of molecular forces, which directly relate to cell function and behavior in their native environment.
Therefore, implementing a molecular force sensor capable of accurately measuring selectin-mediated interactions is crucial to improving our understanding of rolling adhesion. Here, we describe the protocol for the adhesion footprint assay12 where PSGL-1 coated beads are rolled on a surface presenting p-selectin functionalized tension gauge tethers (TGTs)13. These TGTs are irreversible DNA-based force sensors that result in a permanent history of rupture events in the form of fluorescence readout. This is achieved through the rupturing of the TGT (dsDNA) and then subsequent labeling of the ruptured TGT (ssDNA) with a fluorescently labeled complementary strand. One major advantage of this system is its compatibility with both diffraction-limited and super-resolution imaging. The fluorescently labeled complementary strand can either be permanently bound (>12 bp) for diffraction-limited imaging or transiently bound (7-9 bp) for super-resolution imaging through DNA PAINT. This is an ideal system to study rolling adhesion as the TGTs are ruptured during active rolling, but the fluorescence readout is analyzed post-rolling. The two imaging methods also provide the user with more freedom to investigate rolling adhesion. Typically, diffraction-limited imaging is useful for extracting molecular rupture force through fluorescence intensity13, whereas super-resolution imaging allows for quantitative analysis of receptor density. With the ability to investigate these properties of rolling adhesion, this approach provides a promising platform for understanding the force-regulation mechanism on the molecular adhesion of rolling cells under shear flow.
1. Oligonucleotide labeling and hybridization
2. Surface PEGylation
3. Flow chamber preparation
4. Surface preparation
NOTE: Refer to Figure 1B for the overall workflow.
5. Experiment and imaging
The protocol above describes the experimental procedure of the adhesion footprint assay. The general experiment workflow is illustrated in Figure 1, from the flow chamber assembly (Figure 1A) to the surface functionalization (Figure 1B) and experiment and imaging steps (Figure 1C).
Figure 2 is a representative result for the ProtG-ssDNA bioconjugation characterization. The UV/Vis spectra of three components in the final product, namely, ProtG, mal-ssDNA, and imidazole elution buffer, were collected prior to the final conjugation (Figure 2A), each corresponding to a known concentration. These spectra form the orthogonal basis for fitting to the bioconjugation product spectrum, where the three unknown parameters are their concentrations. A custom function in MATLAB was used to determine the concentrations. The results show a nearly 1:1 ratio of ProtG to ssDNA (Figure 2A). This is as expected because the ssDNA has only one amine modification, and the ProtG has a single cysteine engineered at its C-terminus. This approach is more advantageous to the previously reported approach19 using a single thiol modified DNA to target the multitude of primary amines on ProtG, where the conjugation ratio cannot be easily maintained.
Additionally, native PAGE was used to confirm the bioconjugation (Figure 2B). DNA is stained by GelGreen and proteins by Coomassie blue, respectively. As GelGreen stains dsDNA more strongly than ssDNA, it is expected that any ssDNA bands to be dimmer than the equal molar concentration of dsDNA bands (lanes 3, 4). Because the stock ProtG contains a C-terminal cysteine residue, a fraction of the proteins form dimers through a disulfide bond, as seen in lane 5 (Figure 2B). The reduced ProtG, on the other hand, shows a single band (lane 6). When using the stock ProtG in the DNA conjugation directly, the disulfide dimerized ProtG does not react to the DNA and shows as a band without any GelGreen staining (lanes 7, 8). The ProtG dimer band disappears in the conjugation product using reduced ProtG (lanes 9, 10). Because an excess of mal-ssDNA to ProtG (1.5:1) is used during the conjugation, TGT only bands are visible in the final conjugation product (lanes 8, 10). The bright GelGreen bands coinciding with the monomeric ProtG band indicate successful conjugation and good yield.
Figure 3 illustrates representative raw microscopy images and the workflow to correct them for subsequent image-stitching and analysis. The TIRF illumination profile introduced from a single-mode fiber is generally brighter in the middle of the field of view and dimmer around the edges (Figure 3A,B). To compensate for the uneven illumination and flatten the images for quantitative analysis, the illumination profile was determined by averaging thousands of individual frames (Figure 3B). Flattened images were produced by subtracting the camera noise from both raw and illumination profiles and then normalizing by the illumination profile (Figure 3C). The effect of the image flattening is clearly illustrated when the images are stitched to form a large image. Image intensity in the background regions without any cell tracks shows clear periodic patterns corresponding to the uncorrected images (Figure 3D). The same field of view stitched from flattened images produces a flat background (Figure 3E). Having a flat background is critical for interpreting the intensities fluctuations along a cell track. As a first experiment, a ramp-up flow profile similar to the one illustrated in Figure 3F is used to determine the range of shear stress suitable for the experiment ensuring both stable cell rolling and clear fluorescent cell tracks. A typical single-cell adhesion footprint under this flow profile is shown in Figure 3G, where the intensity increases as the shear stress increases until the cell can no longer sustain rolling at high shear stress and detach from the surface, marking the end of a single track.
Figure 4 illustrates potential outcomes from suboptimal to optimal representative experimental results. Figure 4A illustrates a suboptimal outcome where the fluorescent tracks have a low signal-to-noise ratio. This is likely caused by either a low surface density of the fully conjugated probes or a low flow rate. Figure 4B illustrates another suboptimal outcome where the fluorescent tracks are too densely packed to resolve and isolate individual tracks for subsequent analysis. Figure 4C is an example of a good outcome where individual tracks are resolvable over a long distance and clear against the background. Figure 4D is an example of the diffraction-limited TIRF image (left half) in comparison to the same track imaged by DNA-PAINT (right half). The DNA-PAINT in this setup produces a NeNA (nearest-neighbor-based analysis) precision of 28.8 nm.
Figure 1: Experiment workflow of the adhesion footprint assay. (A) Assembly of the flow cell and flow chamber. (B) Surface passivation and functionalization. Each incubation step is marked by the duration, followed by a wash step. (C) Cell rolling experiment and imaging. Cells rolling on the surface will unzip the DNA where adhesion interactions form, leaving ssDNA on the surface that marks the location of each adhesion event. The surface is labeled with the permanent imager ssDNA for extensive area imaging, requiring 2 min staining before washing off. For super-resolution DNA-PAINT imaging, the imager strand is kept in the buffer. Please click here to view a larger version of this figure.
Figure 2: Bioconjugation characterization. (A) UV-VIS absorbance of the Ni-NTA purified bioconjugation product (blue) and the curve fit (red) to determine the conjugation ratio. The absorption spectra of ProtG (magenta), mal-ssDNA (green), and imidazole (gray) were used as components to create the best fit (red) to the product spectrum (blue). The residue of the fit is shown as the black dashed line. This allows us to determine the concentration of ProtG and ssDNA in the purified bioconjugation product and their molar ratio. (B) Native PAGE of components in the bioconjugation procedure. The first lane shows a low molecular weight DNA ladder (25, 50, 75, 100, 150, 200, 250, 300, 350, 500, 766 bp). The gel image is false-colored, with DNA-staining GelGreen in green and Coomassie blue protein stain (inversed) in magenta. Please click here to view a larger version of this figure.
Figure 3: Image processing and representative results from extensive area imaging. (A) Thousands of raw TIRF images tiling an extensive area. (B) Illumination profile derived from the raw images. (C) Corrected images by flattening the illumination profile. (D) Stitched image from raw image tiles. The uneven illumination profile can be seen as periodic patterns in the image. The blue and red boxes indicate the image sections where the mean intensity profiles are projected (blue and red traces). The mean intensity values (arbitrary unit) represent those of 8-bit images (0-255). (E) Same area as (D) but stitched using flattened images. The projections do not show any large-scale periodic patterns. (F) The shear stress profile to use in the experiments to determine the range of shear stresses that result in cell rolling and yield fluorescence tracks. (G) A sample fluorescent track from a single cell under the flow profile illustrated in (F). The cell travels from left to right, the fluorescence intensity increases as the shear stress ramps up until the cell can no longer sustain rolling and detaches from the surface. Please click here to view a larger version of this figure.
Figure 4: Representative suboptimal and optimal results. (A) An example of fluorescent tracks with insufficient contrast. (B) An example of excessive fluorescent track density. (C) An example of optimal track density and contrast. All three images were acquired under the same condition, flattened and stitched. The red boxes in each image represent the area where the intensity projections (right) were taken. (D) A fluorescent track shown in diffraction-limited (left half) and DNA-PAINT (right half) imaging. Please click here to view a larger version of this figure.
Problem | Possible Reason | Solution | |
Epoxy tape not cut properly | Epoxy layer too thick | Thin the epoxy layer as much as possible with the razer blade | |
Laser engraver power and speed not optimized | Optimize laser engraver power and speed | ||
Bubbles stuck on sides of flow chamber | Improper initial liquid introduction | Push buffer solutions through channel at high flow rate to wash the bubbles out | |
Bubbles pass through flow chamber | Improper liquid introduction through inlet and outlet | Ensure a liquid droplet is on the inlet tubing to ensure liquid-to-liquid contact between pipette tip and the inlet | |
Bubbles from the syringe got into the flow line | Tilt the syringe pump to ensure bubbles are trapped at the plunger end | ||
Liquid cannot enter channels | Inlets screwed on too tight | Adjust to optimize seal. The inlet tubing should just touch the channel opening when screwed in properly. | |
Channel leakage | Epoxy hasn’t cured completely | Re-make channels, ensure using 5 min fast curing epoxy and let cure for at least 1 h | |
Inlets not sealing properly | Adjust to optimize seal | ||
Cells stuck | Poor PEGylation leading to non-specific binding | Ideally, remake PEG. Additionally, can try to incubate additional blocking agents (BSA & Tween-20) and add blocking agents to wash buffer. | |
Surface passivation destroyed by large air bubbles passing through the channel | Ensure no bubbles go through channel | ||
Problem with the cells | Use HL-60 2 weeks after restarting cell culture from frozen. Confirm cell rolling on control surface with only P-selectin. | ||
Cells do not or have sparse interactions with the surface | P-selectin density too low, as a result of poor surface functionalization and/or poor bioconjugation | First, use ProtG-biotin instead of ProtG-TGT as a control to determine whether the problem is due to bioconjugation or surface biotin density.After ensuring bioconjugation quality and surface biotin density, increase TGT-ProtG and P-selectin-Fc concentration and incubation time. | |
Cells roll but do not produce fluorescent tracks | Adhesion interactions too weak to rupture TGT | Increase flow rate during rolling experiment to increase the interaction force. We recommend an initial ramp up flow profile (Figure 3F) to find the optimal flow rate that produces both stable rolling and fluorescence tracks (Figure 3G) | |
Bad quality surface leads to high background fluorescence and insufficient contrast to see tracks | Check surface passivation |
Table 1: Troubleshooting. The table lists the possible reasons and solutions for problems occurring when performing this assay
Supplemental Coding File 1: The custom-written MATLAB script to decompose the final product spectrum based on the three spectra collected previously (ProtG, SMCC-strand, Ni-NTA bead elution buffer) to determine the conjugation efficiency and ratio of ProtG to ssDNA. Please click here to download this File.
The adhesion footprint assay enables visualization of the molecular adhesion events between PSGL-1 and P-selectin during cell rolling adhesion. This process is initiated by P-selectin-mediated capturing followed by rolling under fluidic shear stress. Potential issues during the experiment usually involve poor cell rolling or missing fluorescent tracks even when cells roll well. These problems are often resulting from quality controls at the critical steps in the protocol, as listed in the troubleshooting table (Table 1).
Biomolecules and buffers are required to be filtered and stored at 4 °C to prevent contamination because surface preparation involves multiple steps. High-quality surface passivation is a requirement to achieve appropriate surface functionalization density and reduce the nonspecific binding of biomolecules. Nonspecific binding of biomolecules to the surface can create a high fluorescence background, interfering with the single-molecule fluorescence imaging and statistical data analysis. Multiple factors can affect surface passivation. Hydrolysis of aminosilane and PEG-NHS yield much lower efficiency of PEGylation. Sufficient KOH washing and piranha cleaning enhance the hydrophilicity by generating free hydroxyl groups on the glass surface, increasing the density of the chemically reactive group. Cells would be stuck on poorly passivated surfaces. The quality of surface passivation is checked by measuring background fluorescence intensity before and after PEGylation using fluorescent biomolecules.
The surface density of ligands is a critical factor for cell rolling, which is controlled by the PEG: PEG-biotin ratio, TGT hybridization, and P-selectin binding. In this system, a PEG: PEG-biotin ratio of 20:1 is sufficient for surface functionalization with sufficient P-selectin for cell rolling. Efficient TGT hybridization also improves the surface density and the signal-to-noise ratio of the fluorescent tracks. This protocol includes a replenishment step of top-strand-TGT-ProtG to ensure any unhybridized TGT bottom strand is complemented before experiments. Conjugation of DNA to ProtG also affects the surface density. Sulfo-SMCC linker was added to DNA at 10-fold molar excess so that all DNA reacted with the linker. ProtG with a single cysteine residue (ProtG-Cys) at the C-terminus was used to achieve a 1:1 DNA: ProtG conjugation ratio. Because the ProtG-Cys can form dimers through disulfide bonds, treatment of TCEP is needed to reduce the disulfide bond before sulfhydryl-reactive cross-linking reactions. Protein-conjugated DNA and TGT hybridization can be validated with native PAGE analysis, in which conjugate DNA and DNA duplex will show the retarded mobility due to the increase of molecular weight. Assembly efficiency can also be estimated by gel densitometry (Figure 2B). The careful PEGylation and bioconjugation processes are crucial for producing a consistent surface. Occasionally, cell state may affect the cell rolling and track formation. Although PSGL-1 expression over cell density has not been reported, cell density is a potent regulator of the cell cycle and protein expression during the growth phase.
Given that PSGL-1 also functions as a signal transduction receptor and regulates cell proliferation20,21, culture conditions such as cell density are maintained for consistent expression level and binding ability of PSGL-1. Attachment of P-selectin-Fc mediated by ProtG onto the surface is crucial to the adhesion and rolling of cells. The binding kinetics of P-selectin to ProtG is dependent on the concentration. Lower concentrations of P-selectin lead to an increase of time to reach equilibrium. At least 30 min is required for saturation binding for concentrations lower than 100 nM22. 10 nM of P-selectin was used to reduce the nonspecific binding to the surface and increased incubation time for sufficient interaction with ProtG. A 1 h incubation is enough time to induce cell adhesion and rolling in this system.
The TGT and its corresponding force-dependent lifetime is an important factor in the results of this assay. During rolling adhesion, the force on the tether is transmitted through both the TGT and the P-selectin: PSGL-1 interaction. Each of these individual components has a unique force-dependent lifetime, and depending on the applied force, the rupture probability will favor one over the other. For example, it has been shown that when using the TGT described in this article, at forces below 13.6 pN, P-selectin: PSGL-1 primarily dissociates, whereas above 13.6 pN, the TGT primarily dissociates13. This is important to understand when performing this assay because if the shear stress is too low or the beads are rolling too slow, the rupture events will primarily be the P-selectin: PSGL-1 interaction, and there will be minimal or no measurable fluorescence signal from the TGTs. The tension threshold of the TGT will also influence the results. If the TGT ruptures at too high of a force, the rupture events will primarily be P-selectin: PSGL-1, and there will be minimal fluorescence signal.
The method described here allows for the analysis of the molecular rupture forces, as well as the locations of molecular adhesion events involved in rolling adhesion. Instead of real-time detection of adhesion, the most significant advantage of this method is that it allows for post-experiment imaging and analysis. Once the adhesion footprint has been left on the surface in the form of ssDNA, the tracks can still be imaged after 12 h if the flow channels are maintained in a 4 °C fridge in a dark humidity chamber with both inlets and outlets blocked to prevent drying. The interpretation of the fluorescence readout for this assay is dependent on the chosen imaging method. Through super-resolution imaging, this assay achieves high spatial resolution (<50 nm) that allows for quantitative analysis of the density of ruptured TGTs13. The analysis of receptor density or ruptured TGT density would be useful in investigating rolling adhesion behavior under different conditions. Contrarily, diffraction-limited imaging does not provide a high spatial resolution; however, it allows a large surface area to be imaged to analyze the fluorescence tracks of multiple beads over hundreds of fields of view. This is advantageous as the fluorescence intensity of a track can be analyzed for a single bead over a large distance providing information on changes in rolling behavior over time. Such an example is changing the shear stress over time and observing the corresponding changes in fluorescence intensity. Recently, it has been shown that through a simple model, the fluorescence intensity of the tracks can be used to estimate the molecular force distribution13. There is also potential application of ratiometric methods to achieve force quantification with this assay23.
Because cell rolling happens rapidly (10s of µm/s) and over an extended distance (1000s of µm), studying their molecular tension has been challenging with traditional real-time molecular tension sensors. The adhesion footprint assay breaks this demanding constraint to allow for post-event imaging. Although the TGT rupture event does not directly report the magnitude of tension experienced prior to rupture, promising developments have been made in the analysis of the fluorescence tracks to allow for the quantitative investigation of the molecular forces involved in rolling adhesion13,23,24.
The authors have nothing to disclose.
This work was supported by the Canada Foundation of Innovation (CFI 35492), Natural Sciences and Engineering Research Council of Canada Discovery Grant (RGPIN-2017-04407), New Frontiers in Research Fund (NFRFE-2018-00969), Michael Smith Foundation for Health Research (SCH-2020-0559), and the University of British Columbia Eminence Fund.
4-channel drill guide | Custom made | 3D printed with ABS filament | |
4-holes slide | Custom made | Drill clean microscope slide using a Dremel with diamond coated drill bits on a 4-channels drill guide which has a layout that matches with the centers of the 8-32 threaded holes on the aluminum clamp. | |
Acetone | VWR | BDH1101-4LP | |
Acrylic spacer | Custom made | Cut two blocks of acrylic sheets with the dimension of 40 mm x 30 mm x 2.5 mm. On each block, drill two 3 mm holes that are precisely aligned with the 4-40 holes on the aluminium holder. | |
Aluminium chip holder | Custom made | Machine anodized aluminium block into a C-shaped holder with the outer dimension of 640 mm x 500 mm x 65 mm and the opening dimension of 400 mm x 380 mm x 65 mm. Inlets and outlets are tapped with 8-32 thread. | |
Aminosilane | AlfaAesar | L14043 | CAS 1760-24-3 |
Antibiotic/antimycotic solution | Cytiva HyClone | SV3007901 | Pen/Strep/Fungiezone |
Beads, ProtG coated polystyrene | Spherotech | PGP-60-5 | |
Bovine serum albumin | VWR | 332 | |
Buffer, DNA PAINT | 0.05% Tween-20, 5 mM Tris, 75 mM MgCl2, 1 mM EDTA | ||
Buffer, T50M5 | 10mM Tris, 50 mM NaCl, 5 mM MgCl2 | ||
Buffer, Rolling | HBSS with 2mM CaCl2, 2 mM MgCl2, 10 mM Hepes, 0.1% BSA | ||
Buffer, Wash | 10 mM Tris, 50 mM NaCl, 5 mM MgCl2 and 2 mM CaCl2, 0.05% Tween 20 | ||
Calcium chloride | VWR | BDH9224 | |
Cell culture flasks | VWR | 10062-868 | |
Concentrated sulfuric acid | VWR | BDH3072-2.5LG | 95-98% |
Coverslip holding tweezers | Techni-Tool | 758TW150 | |
Diamond-coated drill bits | Abrasive technology | C5250510 | 0.75 mm diamond drill |
DNA, amine-ssDNA (top strand) | IDT DNA | Custom oligo | CCGGGCGACGCAGGAGGG /3AmMO/ |
DNA, biotin-ssDNA (bottom strand) | IDT DNA | Custom oligo | /5BiotinTEG/ TTTTT CCCTCCTGCGTCGCCCGG |
DNA, imager strand for DNA-PAINT | IDT DNA | Custom oligo | GAGGGAAA TT/3Cy3Sp/ |
DNA, imager strand for permanent labelling | IDT DNA | Custom oligo | CCGGGCGACGCAGG /3Cy3Sp/ |
Double-sided tape | Scotch | 237 | 3/4 inch width, permanent double-sided tape |
EDTA | Thermofisher | 15575020 | 0.5 M EDTA, pH 8.0 |
Epoxy | Gorilla | 42001 | 5 minute curing time |
Fetal Bovine Serum (FBS) | Avantor | 97068-085 | |
GelGreen | Biotium | 41005 | |
Glacial acetic acid | VWR | BDH3094-2.5LG | |
Glass, Coverslips | Fisher Scientific | 12-548-5P | |
Glass, Microscope slide | VWR | 48300-026 | 75 mm x 25 mm x 1 mm |
Glass, Staining jar | VWR | 74830-150 | Wheaton Staining Jar (900620) |
Hanks' Balanced Salt solution (HBSS) | Lonza | 04-315Q | |
Hemocytometer | Sigma-Aldrich | Z359629-1EA | |
HL-60 cells | ATCC | CCL-240 | |
Humidity chamber slide support | Custom made | 3D printed with ABS filament | |
Hydrogen peroxide | VWR | BDH7690-1 | 30% |
Imidazole | Sigma-Aldrich | I2399 | |
Inlets/outlets | Custom made | Drill through eight 8-32 set screws using cobalt drill bits. Insert 1.5 cm polyethylene tubing (Tygon, I.D. 1/32” O.D. 3/32”) into each hollow setscrew | |
Iscove Modified Dulbecco Media (IMDM) | Lonza | 12-722F | |
Magnesium chloride | VWR | BDH9244 | |
Magnetic Ni-NTA beads | Invitrogen | 10103D | |
Mailer tubes | EMS | EMS71406-10 | |
Methanol | VWR | BDH1135-4LP | |
Micro Bio-Spin P-6 Gel Columns | Biorad | 7326200 | In SSC Buffer |
PEG | Laysan Bio | MPEG-SVA-5000 | |
PEG-biotin | Laysan Bio | Biotin-PEG-SVA-5000 | |
Potassium hydroxide | VWR | 470302-132 | |
Protein, Protein G | Abcam | ab155724 | N-terminal His-Tag and C-terminal cysteine |
Protein, P-selectin-Fc | R&D System | 137-PS | Recombinant Human P-Selectin/CD62P Fc Chimera Protein, CF |
Protein, Streptavidin | Cedarlane | CL1005-01-5MG | |
Pump Syringe | Harvard Apparatus | 704801 | |
Sodium bicarbonate | Ward’s Science | 470302-444 | |
Sodium chloride | VWR | 97061-274 | |
Sulfo-SMCC | Thermofisher | 22322 | |
Syringe | Hamilton | 81520 | Syringes with PTFE luer lock, 5 mL |
Syringe needles | BD | 305115 | Precision Glide 26 G, 5/8 Inch Length |
TCEP | Sigma-Aldrich | C4706-2G | |
Tris | VWR | BDH4502-500GP | |
Tubing, Adaptor | Tygon | ABW00001 | Formulation 3350, I.D. 1/32”; O.D. 3/32” |
Tubing, Polyethylene | BD Intramedic | 427406 | Intramedic (PE20) I.D. 0.38mm; O.D. 1.09mm |
Tween-20 | Sigma-Aldrich | 93773 |