The authors gratefully acknowledge operational funding from Canadian Institutes of Health Research Project (PTJ 162251), Queen&#39;s University Senate Advisory Research Committee and infrastructure support from the Canadian Foundation for Innovation John Evan&#39;s Leadership Fund (Project 34137) and the Ministry of Research and Innovation Ontario Research Fund (Project 34137). L.A.M. was supported by a Queen&#39;s University R. Samuel McLaughlin Fellowship, a Natural Sciences and Engineering Research Council of Canada Canadian Graduate Scholarship Master&#39;s Award and an Ontario Graduate Scholarship. The authors would like to thank Dr. Myron Szewczuk for his generous gift of the NF-&#954;B/AP-1 reporter macrophage cell line and Drs. Michael Blennerhassett and Sandra Lourenssen for the use of their gel imaging system and plate reader.

1. Media and Reagent Preparation
<ol>
	<li>Prepare fibroblast media. Combine 450 mL of Dulbecco&#39;s modified Eagle medium (DMEM), 50 mL of fetal bovine serum (FBS), and 5 mL of penicillin/streptomycin. Store at 4 &#176;C for up to 3 months.</li>
	<li>Prepare reporter macrophage growth media in 50 mL aliquots. Combine 45 mL of DMEM, 5 mL of FBS, 5 &#956;g/mL mycoplasma elimination reagent (Table of Materials), and 200 &#956;g/mL phleomycin D1 (Table of Materials). Store at 4 &#176;C for up to 3 months.</li>
	<li>Prepare reporter macrophage assay media in 50 mL aliquots. Combine 45 mL of DMEM, 5 mL of heat inactivated FBS (HI-FBS), 5 &#956;g/mL mycoplasma elimination reagent, and 200 &#956;g/mL phleomycin D1. Store at 4 &#176;C for up to 3 months.</li>
</ol>
2. Coating Cell Culture Surfaces with Poly(methyl methacrylate)
<ol>
	<li>Dissolve poly(methyl methacrylate) (PMMA) in chloroform at 20 mg/mL (e.g., 100 mg of PMMA in 5 mL of chloroform) in a 20 mL glass scintillation vial. Place a magnetic stir bar in the vial and allow to stir for at least 2 h, until all solids are dissolved. 
	CAUTION: Chloroform is harmful if inhaled. Ensure to use solvent in a fume hood while wearing PVA gloves.</li>
	<li>Pipette 400 &#181;L of PMMA solution onto the center of a borosilicate glass microscope slide in a spin coater, and spin at 3000 rpm for 2 min. Prepare the number of slides required for the assay, as well as 3&#8722;5 extra for water contact angle measurement. Store slides in a clean box (sprayed and wiped with 70% ethanol) for future use. 
	NOTE: Spin coating is often used to deposit a thin, uniform coating on a flat surface. A spin coater rotates a substrate at high speeds, using centrifugal force to spread the coating solution over the surface.
	<ol>
		<li>Measure water contact angle at two random positions on the surface of extra coated slides (i.e., not the slides being used for cell culture) with a goniometer to ensure glass surface was completely coated with the polymer. 
		NOTE: Only water of the highest purity (e.g., glass triple distilled) should be used for water contact angle measurements.</li>
	</ol>
	</li>
	<li>In a biological safety cabinet (BSC) attach 8-chamber sticky wells to PMMA coated-slides using sterile forceps and following aseptic technique. Press firmly on the top of the sticky wells to make sure they are strongly attached. Incubate the slides with attached sticky-wells at 37 &#176;C overnight to secure the seal.
	<ol>
		<li>Test the seal of the sticky wells by adding 200 &#181;L of cell culture grade (endotoxin-free) water to each well. Incubate at room temperature (RT) for 60 min and ensure no leakage before proceeding. Aspirate the water, being careful not to disturb the PMMA coating.</li>
	</ol>
	</li>
	<li>Perform endotoxin-free water washes by adding 300 &#181;L of endotoxin-free water to each well and incubating for 1 h (three times), 12 h, and 24 h prior to use to remove any remaining solvent.</li>
	<li>Test endotoxin concentration of the slides to be used for cell culture. Incubate 200 &#181;L of endotoxin-free reagent water (Table of Materials) in one well of each slide for 1 h. Measure endotoxin concentration in the extract using an endpoint chromogenic endotoxin assay (Table of Materials). 
	NOTE: The following protocol is specific to the endotoxin assay kit listed in the Table of Materials.</li>
	<li>Use only water and consumables (i.e., pipette tips, microcentrifuge tubes and well plates) that are certified pyrogen-free (i.e., endotoxin-free) for this work. Also, any glassware used in the preparation of the polymer-coated surfaces should be depyrogenated using dry heat sterilization (250 &#176;C for 30 min) prior to use10. Measuring endotoxin in the extract solution, as described here, can result in an underestimation of endotoxin on the material surface11,12. Consequently, it is recommended that when developing a polymer coating protocol, perform the endotoxin assay reaction (i.e., steps 2.5.4&#8722;2.5.6 for test samples [reagent water] or spike controls) directly within wells containing the coated sample to ensure no sources of endotoxin are inadvertently introduced into the system during the coating process.
	<ol>
		<li>Bring all test samples (i.e., extracts) and endotoxin assay reagents to RT. Reconstitute chromogenic reagent in assay buffer and endotoxin standard in reagent water, allow to dissolve for 5 min and gently swirl before using. Cover all bottles with paraffin film when not in use.</li>
		<li>Create a 5&#8722;8 point standard dilution curve of endotoxin standard ranging from the lower to the upper limit of the assay by performing a serial dilution of the endotoxin standard in reagent water.</li>
		<li>To control for enhancement or inhibition of the endotoxin assay in test samples, prepare a positive control (also called a spike control or spiked sample) by diluting a known amount of endotoxin in unused test sample solution. 
		NOTE: The concentration of the positive control should be the same concentration as a standard in the middle of the standard curve. If the recovered amount of the endotoxin spike (i.e., concentration of the positive control minus the concentration of the unspiked test sample) is within 50&#8722;200% of the nominal concentration of the endotoxin spike, the extraction solution can be considered to not significantly interfere with the assay.</li>
		<li>Add 50 &#181;L of standards, samples, or spike controls to each well of a 96-well plate in duplicate or triplicate. Use reagent water as a negative control.</li>
		<li>Add 50 &#181;L of chromogenic reagent to every well. Add reagent quickly to all wells. Use a timer to record the amount of time it takes to add reagent to all wells. Cover the plate with an adhesive seal and incubate at 37 &#176;C (incubation time is lot-dependent and stated on Certificate of Analysis included in the chromogenic reagent kit). Alternatively, check on the plate every 15 min during incubation until color change is observed in all standard wells.</li>
		<li>After incubation, add 25 &#181;L of 50% acetic acid to each well (final concentration of 10% acetic acid per well) to stop the reaction. Add acetic acid in the same order as the chromogenic reagent was added. Read absorbance of the plate using a plate reader at 405 nm. Aspirate liquid and discard plate. 
		NOTE: Acetic acid addition should take the same length of time to add to each well as the chromogenic reagent took (&#177; 30 s).</li>
	</ol>
	</li>
	<li>Ultraviolet (UV) sterilize the slides for 30 min prior to cell culture experiments.</li>
</ol>
3. Coating Cell Culture Surfaces with Polydimethylsiloxane
<ol>
	<li>Mix polydimethylsiloxane (PDMS) elastomer in a 10:1 weight ratio (base:curing agent). In a biological safety cabinet, pipette approximately 10 mL of polydimethylsiloxane base into a sterile tube. Weigh the tube and slowly add curing agent until 10% has been added. 
	CAUTION: Use PDMS reagents in a well-ventilated area and avoid eye contact by wearing safety glasses.</li>
	<li>Thoroughly mix the elastomer by stirring with a sterile serological pipette tip and by pipetting up and down. Add approximately 200 &#956;L of the solution to each well of a 48-well plate. Tilt the well plate slowly to ensure complete coverage of wells with elastomer solution.</li>
	<li>Place the well plate with elastomer into a vacuum oven set at 50 cmHg, 40 &#176;C. Remove the lid and cover with a single-ply wipe to prevent other debris from falling into the wells. Allow to incubate for at least 48 h.
	<ol>
		<li>Confirm the wells are completely coated via visual inspection. Ensure the elastomer is fully cured by gently prodding with a sterile pipette tip before removing.</li>
	</ol>
	</li>
	<li>Add 300 &#181;L of 70% ethanol (made with absolute ethanol and endotoxin-free water) and incubate at RT for 1 h. Remove the ethanol and perform endotoxin-free water washes by adding 300 &#181;L of endotoxin-free water to each well and incubating for 1 h (three times), 12 h, and 24 h prior to use to remove any remaining solvent.
	<ol>
		<li>Incubate 200 &#181;L of endotoxin-free water in three wells of each plate for 1 h. Measure endotoxin concentration of the water extracts using an endpoint chromogenic endotoxin assay (steps 2.5.1&#8722;2.5.6).</li>
	</ol>
	</li>
</ol>
4. Coating Cell Culture Surfaces with Fluorinated Poly(tetrafluoroethylene)
<ol>
	<li>Make a 1 mg/mL solution of fluorinated poly(tetrafluoroethylene) (fPTFE) (e.g., add 10 mg of fPTFE to 10 mL of fluorinated solvent [Table of Materials]) in a 20 mL glass scintillation vial. Place a magnetic stir bar in the vial and allow to stir for at least 24 h, until all solids are dissolved.</li>
	<li>Add approximately 150 &#956;L of the polymer solution to each well of a polystyrene 48-well plate (i.e., not tissue culture treated). Tilt the well plate slowly to ensure complete coverage of all wells with polymer solution. Replace lid.
	<ol>
		<li>To ensure effective fPTFE-coating of wells, glass coverslips should be coated in fPTFE and used for water contact angle measurement (step 4.3.1). Place coverslips inside the wells of a 24-well plate. Add approximately 400 &#956;L of the polymer solution to each well containing a coverslip. Push the coverslips down using sterile forceps, ensuring they are completely covered in polymer solution, and cover the well plate with a lid.</li>
	</ol>
	</li>
	<li>Place the well plate with polymer solution and/or coverslips into a vacuum oven set at 50 cmHg, 40 &#176;C. Remove the lid and cover with a single-ply wipe to prevent other debris from falling into the wells. Allow to incubate for at least 48 h.
	<ol>
		<li>Measure water contact angle of fPTFE-coated coverslips with a goniometer to ensure effective coating. 
		NOTE: Only water of the highest purity (e.g., glass triple distilled) should be used for water contact angle measurements.</li>
	</ol>
	</li>
	<li>Add 300 &#181;L of 70% ethanol (made with absolute ethanol and endotoxin-free water) and incubate at RT for 1 h. Remove the ethanol and perform endotoxin-free water washes by adding 300 &#181;L of endotoxin-free water to each well and incubating for 1 h (three times), 12 h, and 24 h prior to use to remove any remaining solvent.
	<ol>
		<li>Incubate 200 &#181;L of endotoxin-free water in three wells of each plate for 1 h. Measure endotoxin concentration of water extracts using an endpoint chromogenic endotoxin assay (steps 2.5.1&#8722;2.5.6).</li>
	</ol>
	</li>
	<li>UV sterilize the well plates for 30 min prior to cell culture experiments.</li>
</ol>
5. Making Lysate from 3T3 Cells
<ol>
	<li>Grow 3T3 cells in multiple T150 flasks to 70% confluence. To detach cells, aspirate media, wash surface with 5 mL of PBS, and aspirate PBS. Add 5 mL of animal origin-free, recombinant cell dissociation enzyme (Table of Materials) and incubate at 37 &#176;C for 3&#8722;5 min.</li>
	<li>Detach cells by gently tilting the flask back and forth. Add 5 mL of PBS to neutralize the recombinant enzyme used for cell dissociation. Transfer the detached cells from the flasks into a centrifuge tube and mix via pipetting. Perform a live cell count using a hemocytometer and cell viability dye. 
	NOTE: A cell dissociation enzyme that can be neutralized through dilution in PBS was selected to avoid the introduction of serum-based proteins in the lysate preparation. If trypsin is used to dissociate cells, it should be neutralized with a serum-containing solution, and an additional PBS wash should be performed to reduce the amount of serum proteins carried over into the lysate preparation.</li>
	<li>Centrifuge the cells at 200 x g for 5 min. Aspirate the supernatant and resuspend cells in original volume (i.e., 10 mL x number of flasks) of PBS to wash off any remaining media. Repeat.</li>
	<li>Centrifuge the cells again at 200 x g for 5 min and aspirate the supernatant. Add the volume of PBS required to achieve a final cell concentration of 1 x 106 cells/mL. Place the cell solution into a -80 &#176;C freezer until sample is fully frozen (at least 2 h).</li>
	<li>Thaw cell solution in a 37 &#176;C water bath. Once completely thawed, place the solution back into the -80 &#176;C freezer until totally frozen. Repeat for a total of 3 freeze-thaw cycles.</li>
	<li>Perform a micro bicinchoninic acid (BCA) assay on the cell lysate at a variety of dilutions (e.g., 1/100, 1/200, 1/500, 1/1000) to determine the protein concentration. Dilute the cell lysate to a protein concentration of 468.75 &#181;g/mL, aliquot, and store at -80 &#176;C for future use. 
	NOTE: Final protein concentration in a 48-well plate is 125 &#181;g/cm2 (based on the surface area of one well, 0.75 cm2).</li>
	<li>Perform a Western blot to assess presence of DAMPs in lysate (e.g., heat shock protein 60 [HSP60], high mobility group box 1 [HMGB1]) by loading 40&#8722;60 &#181;g of lysate protein in loading buffer onto a 1.5 mm thick 10% polyacrylamide gel and follow standard Western blot procedures.</li>
</ol>
6. Assessing Effect of Adsorbed Protein Layers and Toll-like Receptors on NF-&#954;B Activity of Macrophages
NOTE: For a schematic of the experimental workflow and plate layout, refer to Figure 1A and Supplemental Figure 1, respectively.
<ol>
	<li>Grow reporter macrophages in an appropriately sized flask to 70% confluence. Aspirate media, wash surface with PBS, and aspirate PBS. Add the recombinant cell dissociation enzyme and incubate at 37 &#176;C for 8 min.</li>
	<li>Detach cells by firmly tapping the sides of the flask. Inactivate the recombinant cell dissociation enzyme by adding an equal volume of growth media (containing 10% FBS). Perform a live cell count using a hemocytometer and cell viability dye. 
	NOTE: Expected viability for the reporter macrophages following an 8 min incubation in the cell dissociation enzyme is 90%.</li>
	<li>Centrifuge cells at 200 x g for 5 min. Aspirate supernatant and resuspend in original volume of PBS to wash cells. Centrifuge again and resuspend cells at 7.3 x 105 cells/mL in assay media (containing heat inactivated FBS).</li>
	<li>Separate cell suspension into 3 different tubes: TLR4 inhibitor, anti-TLR2, and untreated. Incubate cells with 1 &#181;g/mL TLR4 inhibitor for 60 min at RT or with 50 &#181;g/mL anti-TLR2 for 30 min at RT.</li>
	<li>Add 200 &#181;L of lysate, 10% FBS, 10% commercial mouse plasma (Table of Materials), or a mixture of the protein solutions to a 48-well plate (or equivalent) and allow protein to adsorb at 37 &#176;C for the desired amount of time (i.e., 30 min, 60 min, or 24 h). Aspirate protein solutions from wells, using a fresh Pasteur pipette for each protein solution, and wash surfaces with 250 &#181;L of PBS for 5 min. Aspirate PBS. Repeat for a total of 3 washes. 
	NOTE: This step may need to be started earlier in the protocol depending on desired adsorption time. Adjust protocol accordingly.</li>
	<li>After incubation period with the TLR4 inhibitor or anti-TLR2, pipette cells to resuspend. Add 200 &#181;L of cell solution to each well.</li>
	<li>For TLR2 positive control condition, add Pam3CSK4 to a final concentration of 150 ng/mL. For TLR4 positive control condition, add lipopolysaccharide (LPS) to a final concentration of 1.5 &#181;g/mL. Incubate cells at 37 &#176;C for 20 h.</li>
	<li>Sample 20 &#181;L of supernatant from each well and plate in duplicate into a 96-well plate. Include three wells of 20 &#181;L assay media as a background control. Add 200 &#181;L of SEAP reporter assay reagent to each well. Cover the plate with an adhesive seal and incubate for 2.5 h at 37 &#176;C. 
	NOTE: The incubation time may vary depending on experimental conditions, and should be optimized for a strong difference in absorbance between positive and negative control wells.
	<ol>
		<li>Transfer the remainder of the supernatant to a 1.5 mL tube (per well). Centrifuge at 1,000 x g for 10 min to pellet any debris. Transfer supernatant to a new 1.5 mL tube and store at -80 &#176;C. Analyze supernatant for the presence of proinflammatory cytokines (e.g., TNF-&#945;, interleukin 6) via enzyme-linked immunosorbent assay (ELISA).</li>
	</ol>
	</li>
	<li>Remove adhesive plate seal. Read absorbance of the plate using a plate reader at 635 nm. Aspirate liquid and discard plate.</li>
</ol>

The authors have nothing to disclose.

A primary focus of our lab is the host response to solid biomaterial soft tissue implants, and in particular how the cellular damage incurred during the implantation procedure impacts the host response. The work presented here describes preliminary experiments using a reporter macrophage cell line and in vitro-generated DAMP-containing cellular lysate, to investigate the influence of molecules released during cellular damage (i.e., from the implant surgery) on macrophage responses to biomaterials. Fibroblast cell lysate ...

The foreign body reaction (FBR) is a chronic host response that can negatively impact the performance of an implanted material or device (e.g., drug delivery devices, biosensors), through the persistent release of inflammatory mediators and by impeding integration between the implanted material and the surrounding tissue1. This innate immune response is initiated by the implantation procedure and is characterized by the long-term presence of innate immune cells and fibrous capsule formation around the implant1. Within the context of material host responses, macrophage-material interactions have a significant impact on the progression of the host response and development of a FBR1. Macrophages are a diverse innate immune cell population, recruited to the implant site either from tissue-resident macrophage populations or from the blood as monocyte-derived macrophages. They begin to accumulate at the implant site shortly after implantation, and within days become the predominant cell population in the implant microenvironment. Material-adherent macrophages, along with foreign body giant cells (FBGC) formed through macrophage fusion, can persist at the material surface for the lifetime of the implant2,3. Consequently, macrophages are considered to be key players in the foreign body response due to their roles orchestrating the characteristic steps of the FBR: acute inflammatory response, tissue remodeling, and formation of fibrotic tissue1.
Toll-like receptors (TLRs) are a family of pattern recognition receptors that are expressed by many immune cells, including macrophages, and have been shown to play a significant role in inflammation and wound healing. In addition to pathogen-derived ligands, TLRs are able to bind endogenous molecules, known as damage-associated molecular patterns (DAMPs), which are released during cell necrosis and activate inflammatory signaling pathways resulting in the production of proinflammatory cytokines4. We and others have proposed that damage incurred during soft tissue biomaterial implantation procedures release DAMPs, which then adsorb to biomaterial surfaces in addition to blood proteins and modulate subsequent cell-material interactions5,6. When macrophages interact with the adsorbed protein layer on an implant, their surface TLRs may recognize adsorbed DAMPs and activate proinflammatory signaling cascades, leading to NF-&#954;B and AP-1 transcription factor activation and production of proinflammatory cytokines. We have previously shown that murine macrophages have significantly increased NF-&#954;B/AP-1 activity and tumor necrosis factor &#945; (TNF-&#945;, proinflammatory cytokine) secretion in response to DAMP-containing adsorbed protein layers on a variety of polymeric surfaces compared to surfaces with adsorbed serum or plasma only (i.e., no DAMPs present), and that this response is largely mediated by TLR2, while TLR4 plays a lesser role5.
The NF-&#954;B/AP-1 reporter macrophage cell line (Table of Materials) used in this protocol is a convenient method to measure relative NF-&#954;B and AP-1 activity in macrophages5,7,8. In combination with TLR pathway inhibitors, this cell line is a useful tool for investigating TLR activation and its role in inflammation in response to a variety of stimuli5,7,8. The reporter cells are a modified mouse macrophage-like cell line that can stably produce secreted embryonic alkaline phosphatase (SEAP) upon NF-&#954;B and AP-1 transcription factor activation9. The colorimetric enzymatic alkaline phosphatase assay (Table of Materials) can then be used to quantify relative amounts of SEAP expression as an indirect measure of NF-&#954;B/AP-1 activity. As NF-&#954;B and AP-1 are downstream of many cell signaling pathways, neutralizing antibodies and inhibitors targeting specific TLRs (e.g., TLR2) or TLR adaptor molecules (e.g., MyD88) can be used to verify the role of a specific pathway. The methodology described in this article provides a simple and rapid approach for assessing the contribution of TLR signaling in murine macrophage responses to a variety of polymeric surfaces with adsorbed protein layers containing both blood proteins and DAMPs as an in vitro model of implanted biomaterials.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Cell culture reagents</td>
		</tr>
		<tr>
			<td>anti-mouse/human CD282 (TLR2)</td>
			<td>Biolegend</td>
			<td>121802</td>
			<td></td>
		</tr>
		<tr>
			<td>CLI-095 (TLR4 inhibitor)</td>
			<td>Invivogen</td>
			<td>TLRL-CLI95</td>
			<td></td>
		</tr>
		<tr>
			<td>C57 complement plasma K2 EDTA 10ml, innovative grade US origin</td>
			<td>InnovativeResearch</td>
			<td>IGMSC57-K2 EDTA-Compl-10ml</td>
			<td>Mouse plasma</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium (DMEM)</td>
			<td>Sigma Aldrich</td>
			<td>D6429-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (DPBS)</td>
			<td>Fisher Scientific</td>
			<td>14190250</td>
			<td>No calcium, no magnesium</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS), research grade</td>
			<td>Wisent</td>
			<td>98150</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS-EK</td>
			<td>Invivogen</td>
			<td>TLRL-EKLPS</td>
			<td>Lipopolysaccharide from Escherichia coli K12</td>
		</tr>
		<tr>
			<td>NIH/3T3 fibroblasts</td>
			<td>ATCC</td>
			<td>CRL-1658</td>
			<td></td>
		</tr>
		<tr>
			<td>Pam3CSK4</td>
			<td>Invivogen</td>
			<td>tlrl-pms</td>
			<td>Synthetic triacylated lipopeptide - TLR1/2 ligand</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Sigma Aldrich</td>
			<td>P4333-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmocin</td>
			<td>Invivogen</td>
			<td>ANT-MPP</td>
			<td>Mycoplasma elimination reagent</td>
		</tr>
		<tr>
			<td>RAW-Blue cells</td>
			<td>Invivogen</td>
			<td>raw-sp</td>
			<td>NF-&#954;B/AP-1 reporter macrophage cell line</td>
		</tr>
		<tr>
			<td>Trypan blue solution, 0.4%</td>
			<td>Fisher Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE express enzyme (1X)</td>
			<td>Fisher Scientific</td>
			<td>12604021</td>
			<td>animal origin-free recombinant cell dissociation enzyme</td>
		</tr>
		<tr>
			<td>Zeocin</td>
			<td>Invivogen</td>
			<td>ANT-ZN-1</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Kits and assays</td>
		</tr>
		<tr>
			<td>ELISA precoated plates, mouse IL-6</td>
			<td>Biolegend</td>
			<td>B213022</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA precoated plates, mouse TNF-&#945;</td>
			<td>Biolegend</td>
			<td>B220233</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotoxin (Escherichia coli) - Control standard endotoxin (CSE)</td>
			<td>Associates of Cape Cope Inc.</td>
			<td>E0005-5</td>
			<td>Endotoxin for standard curve in chromogenic endotoxin assay</td>
		</tr>
		<tr>
			<td>LAL water, 100 mL</td>
			<td>Associates of Cape Cope Inc.</td>
			<td>WP1001</td>
			<td>Used with chromogenic endotoxin assay</td>
		</tr>
		<tr>
			<td>Micro BCA protein assay</td>
			<td>Fisher Scientific</td>
			<td>PI23235</td>
			<td></td>
		</tr>
		<tr>
			<td>Limulus amebocyte lysate (LAL) Pyrochrome endotoxin test kit</td>
			<td>Associates of Cape Cope Inc.</td>
			<td>C1500-5</td>
			<td>Chromogenic endotoxin assay reagent</td>
		</tr>
		<tr>
			<td>QUANTI-Blue alkaline phosphatase detection medium</td>
			<td>Invivogen</td>
			<td>rep-qb2</td>
			<td>Alkaline phosphatase assay to indirectly measure NF-&#954;B/AP-1 activity</td>
		</tr>
		<tr>
			<td colspan="4">Polymeric coating reagents</td>
		</tr>
		<tr>
			<td>Chloroform, anhydrous</td>
			<td>Sigma Aldrich</td>
			<td>288306-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol anhydrous</td>
			<td>Commercial Alcohols</td>
			<td>P006EAAN</td>
			<td>Sigma: Reagent alcohol, anhydrous, 676829-1L</td>
		</tr>
		<tr>
			<td>Straight tapered fine tip forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-113</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorinert FC-40 solvent</td>
			<td>Sigma Aldrich</td>
			<td>F9755-100ML</td>
			<td>Fluorinated solvent for fPTFE</td>
		</tr>
		<tr>
			<td>Cell culture grade water (endotoxin-free)</td>
			<td>Fisher Scientific</td>
			<td>SH30529LS</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(methyl methacrylate) (PMMA)</td>
			<td>Sigma Aldrich</td>
			<td>182230-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184 elastomer kit</td>
			<td>Fisher Scientific</td>
			<td>50822180</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon-AF (fPTFE)</td>
			<td>Sigma Aldrich</td>
			<td>469610-1G</td>
			<td>Poly[4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole-co-tetrafluoroethylene]</td>
		</tr>
		<tr>
			<td colspan="4">Consumables</td>
		</tr>
		<tr>
			<td>Adhesive plate seals</td>
			<td>Fisher Scientific</td>
			<td>AB-0580</td>
			<td></td>
		</tr>
		<tr>
			<td>Axygen microtubes, 1.5 mL</td>
			<td>Fisher Scientific</td>
			<td>14-222-155</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass scintillation vials, with white polypropylene caps</td>
			<td>Fisher Scientific</td>
			<td>03-337-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear PS 48-well plate</td>
			<td>Fisher Scientific</td>
			<td>08-772-52</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear TCPS 96-well plate</td>
			<td>Fisher Scientific</td>
			<td>08-772-2C</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear TCPS 48-well plate</td>
			<td>Fisher Scientific</td>
			<td>08-772-1C</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glasses, circles</td>
			<td>Fisher Scientific</td>
			<td>12-545-81</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tissue culture treated flasks, T25</td>
			<td>Fisher Scientific</td>
			<td>10-126-10</td>
			<td></td>
		</tr>
		<tr>
			<td>sticky-Slide 8 Well</td>
			<td>Ibidi</td>
			<td>80828</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture treated flasks, T150</td>
			<td>Fisher Scientific</td>
			<td>08-772-48</td>
			<td></td>
		</tr>
	</tbody>
</table>

a macrophage reporter cell assay to examine toll-like receptor-mediated nf-kb/ap-1 signaling on adsorbed protein layers on polymeric surfaces

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development and implementation of biomedical devices and tissue engineering constructs. Macrophages, an innate immune cell, are key players in the foreign body reaction because they remain at the implant site for the lifetime of the device, and are commonly studied to gain an understanding of this detrimental host response. Many biomaterials researchers have shown that adsorbed protein layers on implanted materials influence macrophage behavior, and subsequently impact the host response. The methods in this paper describe an in vitro model using adsorbed protein layers containing cellular damage molecules on polymer biomaterial surfaces to assess macrophage responses. An NF-&#1082;B/AP-1 reporter macrophage cell line and the associated colorimetric alkaline phosphatase assay were used as a rapid method to indirectly examine NF-&#1082;B/AP-1 transcription factor activity in response to complex adsorbed protein layers containing blood proteins and damage-associated molecular patterns, as a model of the complex adsorbed protein layers formed on biomaterial surfaces in vivo.

Cleaning methods for the polymer-coated surfaces were tested to ensure there was no disruption of the coating, which would be seen as a change in the water contact angle to an uncoated glass coverslip (Figure 2). Soaking PMMA-coated microscope slides in 70% ethanol for 1 h was found to remove the PMMA coating (Figure 2, left panel), likely due to the solubility of PMMA in 80 wt% ethanol13, therefore PMMA-coated surfaces were cleaned using...

Watch this Scientific Journal Video about A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces at JoVE.com

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

This protocol provides researchers with a rapid, indirect method of measuring TLR-dependent NF-&#1082;B/AP-1 transcription factor activity in a murine macrophage cell line in response to a variety of polymeric surfaces and adsorbed protein layers that model the biomaterial implant microenvironment.

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development ...

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Research

JoVE Journal

Bioengineering

7.3K Views.  Queen's University. This protocol provides researchers with a rapid, indirect method of measuring TLR-dependent NF-кB/AP-1 transcription factor activity in a murine macrophage cell line in response to a variety of polymeric surfaces and adsorbed protein layers that model the biomaterial implant microenvironment.

Video: A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

Lensless Fluorescent Microscopy on a Chip

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for high-throughput screening applications. This emerging technology platform has the potential to eliminate the need for bulky and/or costly optical components through the help of novel theories and digital reconstruction algorithms. Along the same lines, here we demonstrate an on-chip fluorescent microscopy modality that can achieve e.g., &lt;4&mu;m spatial resolution over an ultra-wide field-of-view (FOV) of &gt;0.6-8 cm2 without the use of any lenses, mechanical-scanning or thin-film based interference filters. In this technique, fluorescent excitation is achieved through a prism or hemispherical-glass interface illuminated by an incoherent source. After interacting with the entire object volume, this excitation light is rejected by total-internal-reflection (TIR) process that is occurring at the bottom of the sample micro-fluidic chip. The fluorescent emission from the excited objects is then collected by a fiber-optic faceplate or a taper and is delivered to an optoelectronic sensor array such as a charge-coupled-device (CCD). By using a compressive-sampling based decoding algorithm, the acquired lensfree raw fluorescent images of the sample can be rapidly processed to yield e.g., &lt;4&mu;m resolution over an FOV of &gt;0.6-8 cm2. Moreover, vertically stacked micro-channels that are separated by e.g., 50-100 &mu;m can also be successfully imaged using the same lensfree on-chip microscopy platform, which further increases the overall throughput of this modality. This compact on-chip fluorescent imaging platform, with a rapid compressive decoder behind it, could be rather valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

A lensless on-chip fluorescent microscopy platform is demonstrated that can image fluorescent objects over an ultra-wide field-of-view of e.g., &#62;0.6-8 cm2 with &#60;4&#956;m resolution using a compressive sampling based decoding algorithm. Such a compact and wide-field fluorescent on-chip imaging modality could be valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for ...

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 &mu;m over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lensless Fluorescent Microscopy on a Chip

On-Chip Endothelial Inflammatory Phenotyping

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Cell Patterning on Photolithographically Defined Parylene-C: SiO₂ Substrates

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lensless Fluorescent Microscopy on a Chip

On-Chip Endothelial Inflammatory Phenotyping

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Cell Patterning on Photolithographically Defined Parylene-C: SiO2 Substrates

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Cell Patterning on Photolithographically Defined Parylene-C: SiO₂ Substrates