This project was supported by grants to R.A. from the NIH (AT006885).

All the methods described in this protocol have been approved by the Institutional Animal Care and Use Committee at Wake Forest School of Medicine.
1. Preparation of MCP-1-loaded Growth Factor-reduced Basement Membrane-derived Solution
NOTE: This is a sterile procedure.
<ol>
	<li>Clean cell culture hood with disinfectant before preparing the basement membrane derived solution (e.g., Matrigel).</li>
	<li>Prepare individually packed sterile 1 mL syringe and wipe the top of the basement membrane derived solution vial with an alcohol swab before loading it into the syringe.</li>
	<li>Completely thaw the growth factor-reduced basement membrane-derived solution on ice. 
	CAUTION: If the basement membrane matrix is thawed at room temperature (RT), ensure a small ice particle remains to prevent the solution from gelling.</li>
	<li>Once thawed, open the vial and mix MCP-1 or 1xPBS with 0.1% BSA in the cell culture hood.</li>
	<li>Prepare a 1 mL of the sterile syringe with a 26 G needle and cool on ice.</li>
	<li>Prepare a stock solution of MCP-1 by dissolving MCP-1 to a final concentration of 50 &#181;g/mL in 1x PBS with 0.1% BSA.</li>
	<li>Dilute MCP-1 in 5 mL cold basement membrane-derived solution to a final concentration of 500 ng/mL. Prepare an equal amount of basement membrane-derived solution with vehicle only (1x PBS contain 0.1% BSA).</li>
	<li>Load 500 &#181;L of basement membrane-derived solution (with or without MCP-1) in the cold 1 mL syringe.</li>
	<li>Remove all air bubbles from the basement membrane-derived solution-loaded syringe before making the injection to ensure a uniform plug formation.</li>
</ol>
2. Injection of Basement Membrane-derived Solution
<ol>
	<li>Spray 70% ethanol around the procedure area before and during the injections to disinfect the environment.</li>
	<li>To induce anesthesia, place the mouse in an anesthesia chamber and set the O2 flow meter to 1 liter/min then adjust the vaporizer to 2% isoflurane.</li>
	<li>Monitor the depth of anesthesia by monitoring the respiratory rate (rate/min), corneal reflex and movement of whiskers.</li>
	<li>During the whole procedure, maintain anesthesia using a nose cone.</li>
	<li>During the procedure keep monitoring the respiratory rate (rate/min), corneal reflex and movement of whiskers to verify the depth of anesthesia.</li>
	<li>After confirming lack of response to toe pinch, slowly inject (approximately 100 &#956;L/s) the basement membrane-derived solution subcutaneously into the right (no MCP-1) and left (with MCP-1) flank of the mouse, respectively. If the injection is too slow, the basement membrane-derived gel plugs can become irregularly shaped, or even fragmented, and difficult to remove.</li>
	<li>Hold the syringe for 20-30 s after the injection to prevent the leakage of the solution and to allow a single smooth gel-plug to form.</li>
	<li>After injection, placing the mouse on the warming pad with monitoring until recovery.</li>
	<li>Return the mouse to the original cage once the mouse is fully recovered.</li>
</ol>
3. Harvest the Basement Membrane-derived Gel Plugs
<ol>
	<li>Weigh two separate 1.5 mL microcentrifuge tubes for each mouse and label them.</li>
	<li>Three days after injection of the basement membrane-derived solution, euthanize the mouse in the anesthetic chamber using 3% isoflurane followed by cervical dislocation.</li>
	<li>Remove the mouse&#39;s dorsal fur using hair clippers or apply hair remover cream with cotton-tipped sticks for 5 min and then wipe off.</li>
	<li>Hold the mouse skin using forceps around the lower thoracic and upper lumbar vertebra and make a 2 mm-long incision into the skin.</li>
	<li>Cut the midline of the back of the mouse from an incision made up to cervical vertebrae and down to 1 cm above caudal vertebra junction (tail junction) using surgical scissors.</li>
	<li>Separate the skin from the muscle layer and pin-down the skin on a polystyrene foam platform.</li>
	<li>Carefully hold the fibrous capsule that contains the basement membrane-derived gel plug using fine forceps and remove the fibrous capsule using fine scissors to clean the plug.</li>
	<li>Remove the cleaned basement membrane-derived gel plug and transfer to 1.5 mL microcentrifuge (see 3.1).</li>
	<li>Weigh the microcentrifuge tube with the cleaned basement membrane-derived gel plug and calculate the weight of the basement membrane-derived gel plug.</li>
</ol>
4. Digest the Basement Membrane-derived Gel Plugs
<ol>
	<li>Thaw dispase (10 mL) on the ice.</li>
	<li>Mince the basement membrane-derived gel plug in the microcentrifuge tube using clean fine scissors.</li>
	<li>Add 800 &#181;L of dispase into each microcentrifuge tube containing basement membrane-derived gel plug.</li>
	<li>Vortex with maximum speed for 10 s to disrupt the plug.</li>
	<li>Incubate the microcentrifuge tubes for 2 h at 37 &#176;C at 1,400 rpm in a thermomixer to completely dissolve the basement membrane-derived gel plug.</li>
	<li>After 2 h, centrifuge at 400 x g for 10 min at RT. Carefully remove and discard the supernatant.</li>
	<li>Resuspend the pellet in 1 mL of 1x PBS by inverting or tapping.</li>
	<li>Repeat the centrifugation at 200 x g for 10 min at RT. Carefully remove and discard the supernatant.</li>
	<li>Resuspend the pellet in 300 &#181;L of PBS.</li>
	<li>Take 50 &#181;L of cell suspension in a separate microcentrifuge tube.</li>
	<li>Add 0.5 &#181;L of calcein AM (1 M) to the cell suspension. 
	NOTE: Calcein AM is a green fluorescent cell viability dye which only accumulates in living cells.</li>
	<li>Incubate the cells in a CO2 incubator at 37 &#176;C for 10 min.</li>
	<li>Count green fluorescent (live) and non-fluorescent (dead) cells using an automated cell counter.</li>
</ol>
5. Determining Cell Composition in the Cell Suspension Using Single-cell Western Blot (scWB) Analysis
<ol>
	<li>Rehydrate the scWB chip before use in 15 mL of 1x suspension buffer in a Petri dish for at least 10 min at RT.</li>
	<li>Add 1 mL of the diluted single cell suspension (10,000-100,000 cells/mL) to the rehydrated chip. Set the surface onto the bottom of a 10 cm Petri dish. Ensure that the surface is set flat.</li>
	<li>Cover the Petri dish with a lid to prevent drying and let the cells settle for 5-15 min by gradient.</li>
	<li>Place the chip under a bright-field microscope and inspect the wells at 10x magnification. 
	NOTE: Approximately 15-20% microwells should be occupied by a single-cell, fewer than 2% of wells should contain 2 or more cells</li>
	<li>Tilt the 10 cm Petri dish containing the chip by 45 degrees.</li>
	<li>Wash the chip with the 1x suspension buffer to remove uncaptured cells from the surface of the chip by gentle pipetting from top to bottom of the chip. Repeat 3 times.</li>
	<li>Prepare the single cell Western instrument.</li>
	<li>Set the lysis time, electrophoresis time, and UV capture time. To analyze recruited macrophages recovered from the basement membrane-derived gel plug, use the following settings: lysis time: 0 s; electrophoresis time: 160 s; UV capture time 240 s.</li>
	<li>Carefully load the chip into the electrophoresis cell of single cell Western instrument, gel-side facing. Use caution not to damage the gel side of the chip.</li>
	<li>Pour the lysis/running buffer into the chamber and completely cover the entire single cell Western chip. Start the cell lysis.</li>
	<li>Run scWB instrument using the setting listed in 5.8.</li>
	<li>Once the run is complete, transfer the chip to a 10 cm Petri dish and wash the chip twice with 1x washing buffer for 10 min at RT.</li>
	<li>Prepare dilutions of primary antibody (anti-vinculin: 1:10; anti-CD45: 1:15, anti-CD11b: 1:20, anti-F4/80: 1:10) in a total volume of 80 &#181;L of antibody diluent (mix 8 &#181;L of antibody 1 plus 8 &#181;L of antibody 2 plus 64 &#181;L of diluent).</li>
	<li>Add 80 &#181;L of the primary antibody solution to the antibody probing chamber and lower the chip gel-side down so that the antibody solution wicks across the chip.</li>
	<li>Incubate with the primary antibody solution for 2 h at RT.</li>
	<li>Wash the chip three times for 10 min in 1x washing buffer on the shaker.</li>
	<li>Wash the chip once for 10 min in the water on the shaker to desalt the gel.</li>
	<li>Prepare a 1:20 dilution of secondary antibody in a total volume of 80 &#181;L (mix 2 &#181;L of secondary antibody plus 78 &#181;L of diluent).</li>
	<li>Incubate the chip with secondary antibody for 1 h at RT protected from light.</li>
	<li>Wash the chip three times for 10 min with 1x washing buffer on the shaker.</li>
	<li>Spin the chip using a slide spinner to remove any remaining washing buffer.</li>
	<li>Scan the chip on a dual-laser microarray scanner at 5 &#181;m resolution at the spectral channel of the fluorophore coupled to the secondary antibodies.</li>
	<li>Analyze data using scWB-specific software.</li>
</ol>
6. Stripping the scWB Chip
<ol>
	<li>Store the scanned chip in wash buffer until ready to strip.</li>
	<li>Place the water bath in a fume hood.</li>
	<li>Place a 50 mL tube rack in the water bath with the water just 1 cm above the rack and set the water temperature at 60 &#176;C.</li>
	<li>Prepare the stripping buffer. For 1 L of stripping buffer solution, dissolve 9.85 g of Tris-HCl (pH 6.8) and 20 g of SDS in 900 mL of distilled water and adjust pH. Then fill with distilled water to 1L. Add 0.8% (v/v) of &#946;-mercaptoethanol (&#946;-ME; 14.3 M) immediately before each use.</li>
	<li>After the initial scan, place the chip in a 10 cm Petri dish before stripping.</li>
	<li>For each chip, take 40 mL of stripping buffer and add 320 &#181;l of &#946;-ME. 
	CAUTION: &#946;-ME is toxic and hazardous to human and environment. The following procedure should be done in a fume hood.</li>
	<li>Place the chip in the canister and seal the canister with parafilm to prevent water leaking into the canister.</li>
	<li>Place the canister inside the tube rack in the pre-warmed water bath.</li>
	<li>Incubate for 90 min.</li>
	<li>Carefully remove the chip from the canister and place in a fresh Petri dish.</li>
	<li>Briefly wash the chip once with 1x wash buffer. Then add 15 mL of 1x washing buffer to the Petri dish.</li>
	<li>Wash the chip for 15 min on a shaker. Repeat the wash step four times. 
	NOTE: Chip is ready for the next primary antibody (see steps 5.12 - 5.21).</li>
</ol>

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Open Access fees for this article were provided by ProteinSimple, a Bio-Techne brand.

We developed an in vivo chemotaxis assay, making use of the unique physical properties of the basement membrane-derived matrix and the ability to load this unique matrix with chemokines and inject it into mice. The assay allows us to assess in living mice the responsiveness of monocytes (and macrophages) to chemokines, as illustrated here for MCP-1, and the effects of either genetic manipulation or pharmacological, dietary and other environmental exposures on monocyte chemotaxis. Because the chemokine gradient we create in these mice is essentially unaffected by genetic, pharmacological, dietary or environmental exposure, changes in monocyte chemotactic activity actually reflect functional changes in these monocytes and, as we showed previously, also the reprogramming at both the protein and transcriptional level17,20. We reported that metabolic stress in mice increases monocyte responsiveness to chemoattractant and that this hyper-chemotactic activity correlates with increased protein S-glutathionylation, a reversible, redox-dependent posttranslational protein modification, which in most cases leads to loss of enzymatic and protein function21,22, and in some instances even promotes protein degradation16,23.
To successfully execute this procedure and to obtain optimal results with this assay it is critical that accurate volumes of basement membrane-derived solution are injected slowly to create a singular well-formed plug and fibrous capsules should be removed completely to get clean plugs when harvest basement membrane-derived gel plugs, facilitates the dissolution of the entire plug in the dispase solution. Our data show that leaving the plug in the animals for three days optimizes 1) the signal intensity, i.e., the number of monocytes and macrophages recruited into each plug, 2) the selectivity of the signal, i.e., the content of monocytes and macrophages within the total cell populations and 3) the specificity of the signal, i.e. the increase in monocytes and macrophages in response to MCP-1 compared to vehicle. Finally, we demonstrate how state-of-the-art single cell-based approaches in conjunction with the basement membrane-derived gel plug assay can be used to characterize the monocytes recruited into the plugs and thus obtain a snapshot of the potential phenotype of the monocyte-derived macrophages being recruited to sites of injury in any given mouse model in response to specific genetic, pharmacological, dietary or environmental interventions.
There are a number of limitations of the assay to be considered. First, mouse handling, including anesthesia, injection of basement membrane-derived solution, and surgical dissection requires practice to generate single plugs and reproducible data. Second, while most cells recruited into the MCP-1 loaded plugs are monocytes and macrophages, a substantial number are not. This may affect the interpretation of other, non-single-cell based analytical assays performed on this cell population. Finally, since the cell lysis conditions for scWB are mild, migration distances of proteins may differ from standard WB and may not correlate with protein size. Therefore, both cell lysis and run times have to be validated for each new protein to be tested and each primary antibody used.

The recruitment of monocyte-derived macrophages is essential for the development of chronic inflammatory diseases associated with metabolic disorders, including atherosclerosis and obesity1,2,3,4. The number of monocyte-derived macrophages at sites of tissue injury as well as their plasticity are critical for the tissue homeostasis and repair. Both under- and over-recruitment of monocyte-derived macrophages can impair wound healing5. Triggering local tissue inflammation, for example, by the accumulation and oxidation of low-density lipoprotein (LDL) in the aortic wall or inflammatory activation of adipocytes by fatty acids or bacterial lipopolysaccharide leaking through the intestinal barrier, leads to the release of inflammatory mediators, including chemokines such as monocyte chemoattractant protein-1 (MCP-1/CCL2). MCP-1 is a member of the C-C chemokine family and a key chemoattractant responsible for the recruitment of monocyte-derived macrophages to sites of tissue inflammation and injury6,7,8,9,10. Inflammatory activation of vascular endothelium results in the expression of adhesions molecules such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1)11,12, allowing circulating monocytes activated by MCP-1 to roll along, firmly adhere to and subsequently transmigrate into the subendothelial space12. Infiltrating monocytes differentiate into macrophages, which can be activated into a proinflammatory phenotype, driving the acute inflammatory process. Driven by the microenvironment, pro-inflammatory macrophages can convert into inflammation-resolving macrophages, which play critical roles in the clearing of inflammatory cells, removing pro-inflammatory signals and completing tissue repair and wound healing12,13.
Chronic metabolic stress primes monocytes for dramatically enhanced responsiveness to chemo-attractants and increased monocyte recruitment, and we showed that primed monocytes give rise to macrophages with dysregulated activation programs and polarization states14,15,16. Monocyte priming promotes atherosclerosis, obesity, and possibly other chronic inflammatory diseases associated with metabolic disorders such as steatohepatitis, kidney diseases and possibly cancer. To assess and quantify monocyte priming in mouse models of human diseases, we developed a new technique to assess the priming state of blood monocytes by measuring the chemotactic activity in vivo14,15,16,17. Our approach involves the injection of basement membrane-derived gel loaded with either a chemoattractant &#8212; we commonly use MCP-1 &#8212; or vehicle into the left and right flank, respectively, of mice. When carefully injected subcutaneously, the basement membrane-derived gel will form a single plug, from which the chemokines can diffuse and create a defined chemotactic gradient that is not affected by the metabolic or inflammatory state of the surrounding tissue or the recipient mouse.
The basement membrane-derived gel we use for our assay is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in such extracellular matrix (ECM) proteins. This complex protein mixture contains laminin (60%), collagen IV (30%), the bridging molecule entactin (8%), and a number of growth factors. The basement membrane matrix plug assay was originally developed to investigate angiogenesis in response to various growth factors18,19. However, in order to study monocyte chemotaxis, it is important to use growth factor-depleted basement membrane-derived gel to minimize endothelial cell recruitment and angiogenesis. What makes Matrigel (referred to as basement membrane-derived gel henceforth) unique and particularly useful is that at temperatures below 10 &#176;C it liquefies, allowing for chemokines to be dissolved. At temperatures above 22 &#176;C, the basement membrane-derived solution rapidly undergoes a phase transition and rapidly forms a hydrogel. The plugs can be surgically excised, cleaned and dissolved with a bacillus-derived neutral metalloprotease to yield single cell suspensions, which can be analyzed on a fluorescence-activated cell sorter (FACS), by RNAseq, single-cell RNA and a variety of other omics techniques. Here we describe the use of single-cell Western blot analysis for the characterization of the cell populations recruited into the basement membrane-derived gel plugs one, three or five days after injection.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">10 cm petri dish</td>
			<td style="width:103px;">griner bio-one</td>
			<td style="width:123px;">664 160</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">10x suspension buffer</td>
			<td style="width:103px;">proteinsimple</td>
			<td style="width:123px;">R101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">15 cm petri dish</td>
			<td style="width:103px;">Falcon</td>
			<td style="width:123px;">353025</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">2-Mercaptoethanol</td>
			<td style="width:103px;">MP BIOMEDICALS</td>
			<td style="width:123px;">2194705</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">5% washing buffer</td>
			<td style="width:103px;">proteinsimple</td>
			<td style="width:123px;">R252</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Antibody diluent</td>
			<td style="width:103px;">proteinsimple</td>
			<td style="width:123px;">042-203</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Bench Top Centrifuge</td>
			<td style="width:103px;">Beckman Coulter</td>
			<td style="width:123px;"></td>
			<td>Microfuge 22R Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Bovine Serim Albumin</td>
			<td style="width:103px;">Sigma-Aldrich</td>
			<td style="width:123px;">A7906-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Calcein AM</td>
			<td style="width:103px;">ThermoFisher</td>
			<td style="width:123px;">C3099</td>
			<td>1 ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">CD11b</td>
			<td style="width:103px;">Novus Biologicals</td>
			<td style="width:123px;">NB110-89474</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">CD45 (D4H7K) rabbit mAb</td>
			<td style="width:103px;">Cell Signal Technologies</td>
			<td style="width:123px;">727987S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">CD68/SR-D1 (FA-11)</td>
			<td style="width:103px;">Novus Biologicals</td>
			<td style="width:123px;">NBP2-33337SS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Cellometer Vision</td>
			<td style="width:103px;">Nexcelom</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Dispase</td>
			<td style="width:103px;">BD</td>
			<td style="width:123px;">354235</td>
			<td>100 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Dissecting sissor</td>
			<td style="width:103px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Donkey anti-Rabbit IgG secondary antibody [NL557]</td>
			<td style="width:103px;">Novus Biologicals</td>
			<td style="width:123px;">NL004</td>
			<td>NL 557 conjugate</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:219px;">Donkey anti-Rat IgG (H+L) secondary antibody [DyLight 650]</td>
			<td style="width:103px;">Novus Biologicals</td>
			<td style="width:123px;">NBP1-75655</td>
			<td>DyLight 650 conjugate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">F4/80 (CI-A3-1)</td>
			<td style="width:103px;">Novus Biologicals</td>
			<td style="width:123px;">NB600-404SS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Heat Block</td>
			<td style="width:103px;">effendorf</td>
			<td style="width:123px;">22331</td>
			<td>Thermomixer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Lysis/Running buffer</td>
			<td style="width:103px;">proteinsimple</td>
			<td style="width:123px;">R200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Matrigel Matrix (Grwoth Factor Reduced)</td>
			<td style="width:103px;">BD</td>
			<td style="width:123px;">354230</td>
			<td>10 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Microarray scanner</td>
			<td style="width:103px;">PerkinElmer</td>
			<td style="width:123px;">ScanArray Gx</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Microcentrifuge tube</td>
			<td style="width:103px;">Fisher Scientific</td>
			<td style="width:123px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Microscope</td>
			<td style="width:103px;">ThermoFisher</td>
			<td style="width:123px;">Evos fl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Milo</td>
			<td style="width:103px;">proteinsimple</td>
			<td style="width:123px;"></td>
			<td>Single cell western</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Needle</td>
			<td style="width:103px;">BD</td>
			<td style="width:123px;">305111</td>
			<td>26G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Parafilm</td>
			<td style="width:103px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Probing chamber</td>
			<td style="width:103px;">proteinsimple</td>
			<td style="width:123px;">035-020</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Recombinant mouse CCL2/JE/MCP-1 protein</td>
			<td style="width:103px;">R&#38;D</td>
			<td style="width:123px;">479-JE-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">scWEST chip</td>
			<td style="width:103px;">proteinsimple</td>
			<td style="width:123px;">C300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Shaker</td>
			<td style="width:103px;">Bioexpress</td>
			<td style="width:123px;"></td>
			<td>Gene mate orbital shaker</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Single cell western chip canister</td>
			<td style="width:103px;">proteinsimple</td>
			<td style="width:123px;">035-118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Slide spinner</td>
			<td style="width:103px;">Labnet</td>
			<td style="width:123px;">C1303-T</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Sodium Dodecyl Sulfate (SDS)</td>
			<td style="width:103px;">Fisher Scientific</td>
			<td style="width:123px;">BP 166-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Syringe</td>
			<td style="width:103px;">BD</td>
			<td style="width:123px;">309602</td>
			<td>1 ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Tris-Hydrochloride</td>
			<td style="width:103px;">Fisher Scientific</td>
			<td style="width:123px;">BP 153-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Tweezer</td>
			<td style="width:103px;">proteinsimple</td>
			<td style="width:123px;">035-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Water bath</td>
			<td style="width:103px;">ThermoFisher</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of monocyte chemotactic activity in vivo and characterization of blood monocyte derived macrophages

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of monocyte-derived macrophages can impair wound healing. We showed that high fat and high sugar diets promote monocyte priming and dysfunction, converting healthy blood monocytes into a hyper chemotactic phenotype poised to differentiate into macrophages with dysregulated activation profiles and impaired phenotypic plasticity. The over-recruitment of monocyte-derived macrophages and recruitment of macrophages with dysregulated activation profiles is believed to be a major contributor to the development of chronic inflammatory diseases associated with metabolic disorders, including atherosclerosis and obesity. The goal of this protocol is to quantify the chemotactic activity of blood monocytes as a biomarker for monocyte priming and dysfunction and to characterize the macrophage phenotype blood monocytes are poised to differentiate into in these mouse models. Using single cell Western blot analysis, we show that after 24 h 33%of cells recruited into MCP-1-loaded basement membrane-derived gel plugs injected into mice are monocytes and macrophages; 58% after day 3. However, on day 5, monocyte and macrophage numbers were significantly decreased. Finally, we show that this assays also allows for the isolation of live macrophages from the surgically retrieved basement membrane-derived gel plugs, which can then be subjected to subsequent characterization by single cell Western blot analysis.

To access the responsiveness of blood monocytes to chemoattractants, we injected vehicle-loaded and MCP-1 loaded basement membrane-derived solution into the left and right flanks of each mouse. Basement membrane-derived gel plugs were removed 1, 3 and 5 days after injections, dissolved and cells recruited into each plug were counted (Figure 1A). By subtracting the cell count in the vehicle-loaded plug (open bars) from the cell count in the MCP-1 loaded plug (closed bars), we obtained the number of cells specifically recruited in response to MCP-1 (&#916;&#160;Cell Number, Figure 1B). We observed an accelerated MCP-1-specific recruitment and accumulation of cells over the 5-day period, with rates increasing from 31,000 cells/day (Day 1) to 78,000 cells/day (Day 3) and 136,000 cells/day at day 5 (Figure 1B).
To identify the cell types that entered the basement membrane-derived gel plugs, we subjected the cells isolated from each plug to single cell Western blot analysis (scWB), probing for CD45, CD11b and F4/80 expression to identify monocytes (CD11b+F4/80-), macrophages (CD11b+F4/80+ plus CD11b-F4/80+) and remaining non-monocytic leukocytes (CD45+CD11b-F4/80-) (Figure 2). The scWB chips were then probed for vinculin to determine the total cell numbers on each gel and to confirm single cell occupancy of the microwells on the scWB chip. The percentage of monocytes within the MCP-1-loaded basement membrane-derived gel plugs was low at day 1, 20%, peaked at day 3 at 47%, respectively before dropping to 14% at day 5 (Figure 2D). Macrophage numbers within the MCP-1-loaded basement membrane-derived gel plugs increased steadily from 13% at day 1, to 22% on day 3 and 23% on day 5. For MCP-1-loaded plugs, the percentage of monocytes plus macrophages within the isolated cell population was highest at day 3, 31% in vehicle loaded plugs (Figure 3A)&#160;and 58% in MCP-1-loaded plugs (Figure 3B), indicating that after 3 days the vast majority of cells recruited by MCP-1-dependent chemotaxis are monocytes and macrophages. Even though the total number of monocytes plus macrophages was higher at day 5 compared to day 3 (Figure 3C and D), due to the significantly higher proportion of monocytes and macrophages in the total cell population (Figure 3A and B), the cell count at day 3 more accurately reflects blood monocyte chemotaxis and recruitment. We, therefore, routinely inject the basement membrane-derived solution three days prior to sacrificing the animals. Whether all macrophages recruited by MCP-1 are monocyte-derived or resident macrophages recruited from neighboring tissues contributed was not determined.
Statistical analysis of the data shown here was conducted with analysis software (e.g., SigmaPlot 14). ANOVA followed by the Fischer LSD test was used to compare mean values between experimental groups. All data are presented as mean &#177; SD of at least 3 independent experiments. Results were considered statistically significant at the P &#60; 0.05 level.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59706/59706fig1v2.jpg" /> 
Figure 1:&#160;Quantitation of cells recruited into subcutaneous basement membrane-derived gel plugs. (A) Absolute cell numbers for vehicle-loaded (open bars) and MCP-1-loaded basement membrane-derived gel plugs (closed bars). (B) The number of cells recruited into basement membrane-derived gel plugs by MCP-1 calculated as the difference in cell numbers between MCP-1-loaded and vehicle-loaded plugs. Results are shown as mean &#177; SD (n=4) <a href="https://www.jove.com/files/ftp_upload/59706/59706fig1v2large.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/59706/59706fig2.jpg" /> 
Figure 2:&#160;Analysis of cell populations recruited into basement membrane-derived gel plugs by single cell western blot analysis. (A+B) Representative examples are shown for the scWB analysis of a vehicle-loaded (Control) and an MCP-1-leaded plug (MCP-1) isolated from a mouse three days after injection. Chips labeled with antibodies directed against CD45, CD11b and F4/80 followed by fluorescent secondary antibodies as described in the Protocol. The fluorescence intensity of the labeled band for each individual cell is shown as peak area. (C+D) Cell populations were identified as monocytes (CD11b+F4/80-, <img alt="Image 1" src="/files/ftp_upload/59706/59706img01.jpg" />), macrophages (CD11b+F4/80+ plus CD11b-F4/80+, <img alt="Image 2" src="/files/ftp_upload/59706/59706img02.jpg" style="font-size: 14px;" />) or as non-monocytic leukocytes (CD45+, <img alt="Image 3" src="/files/ftp_upload/59706/59706img03.jpg" style="font-size: 14px;" />). The remaining cells were labeled &#34;other&#34; (<img alt="Image 4" src="/files/ftp_upload/59706/59706img04.jpg" style="font-size: 14px;" />). Results are shown as mean &#177; SD (n = 3). <a href="https://www.jove.com/files/ftp_upload/59706/59706fig2large.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/59706/59706fig3.jpg" /> 
Figure 3:&#160;Monocyte and macrophage content of basement membrane-derived gel plugs. Quantitative analysis of recruited monocyte and macrophage content in vehicle-loaded and MCP-1-loaded plugs removed at day 1, 3 and 5 post-injection. Values are shown as percent of the total cell population (A + B) and in absolute cell numbers per plug (C + D). Results are shown as mean &#177; SD (n = 3). <a href="https://www.jove.com/files/ftp_upload/59706/59706fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages at JoVE.com

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Here we present a protocol to quantify the chemotactic activity of blood monocytes in mouse models, to assess the effects of nutritional, pharmacological and genetic interventions on blood monocyte and to characterize the blood monocytes derived macrophages in mouse models using monocyte-chemoattractant protein-1 (MCP-1)-loaded basement membrane-derived gel plugs.

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of ...

quantification-monocyte-chemotactic-activity-vivo-characterization

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JoVE Journal

Immunology and Infection

9.5K Views.  Wake Forest School of Medicine. Here we present a protocol to quantify the chemotactic activity of blood monocytes in mouse models, to assess the effects of nutritional, pharmacological and genetic interventions on blood monocyte and to characterize the blood monocytes derived macrophages in mouse models using monocyte-chemoattractant protein-1 (MCP-1)-loaded basement membrane-derived gel plugs.

Video: Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&amp;#946; in Human Monocyte-derived Dendritic Cells

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining.

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic ...

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1&#946; and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1&#946; release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...