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.

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

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Immunology and Infection

9.6K Views.  Wake Forest School of Medicine. Deze methodologie stelt ons in staat om de chemotactische activiteit van bloedmonocyt bij levende dieren te kwantificeren en het effect van slechte diëten op monocytenfunctie en hun onderliggende mechanismen te bestuderen. Monocyten-afgeleide macrofagen kunnen worden geïsoleerd en geanalyseerd met behulp van verschillende benaderingen, waaronder eencellige, en omics technieken die een momentopname van de gezondheid van de bloed monocyt site in muismodellen. De procedure van vandaag zal word...

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

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

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 ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the transmigration of innate immune monocytes to inflammatory sites of deposited lipid called fatty streaks, which are present in arterial walls of medium to large arteries. The key pathogenic feature of lesions at this early stage of atherosclerosis is the maturation of monocytes which migrate into arteries to form foam cells or lipid-laden macrophages. Considerable evidence supports the hypothesis that risk of atherosclerosis is increased by chronic inflammatory conditions accompanying diseases such as rheumatoid arthritis and HIV, as well as general ageing, and that this risk is predicted by monocyte activation. While mouse models provide a good platform to investigate the role of monocytes in atherogenesis in vivo, they require genetic alteration of natural cholesterol metabolism and drastic alteration of normal mouse diets, and have limited suitability for the study of atherogenic influences of human comorbid diseases. This motivated us to develop a human in vitro model to measure the atherogenic potential of monocytes isolated from individuals with defined disease states. Currently, human in vitro models are limiting in that they evaluate monocyte transmigration and foam cell formation in isolation. Here we describe a protocol in which monocytes isolated from patient blood transmigrate across human endothelial cells into a type 1 collagen matrix, and their propensity to mature into foam cells in the presence or absence of exogenous lipid is measured. The protocol has been validated for the use of human monocytes purified from individuals with HIV infection and elderly HIV uninfected individuals. This model is versatile and allows monocyte transmigration and foam cell formation to be evaluated using either microscopy or flow cytometry as well as allowing the assessment of atherogenic factors present in serum or plasma.

We describe a protocol to measure transmigration by monocytes across human endothelial monolayers and their subsequent maturation into foam cells. This provides a versatile method to assess the atherogenic properties of monocytes isolated from people with different disease conditions and to evaluate factors in blood which may enhance this propensity.

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the ...

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development and autoimmune diseases. This is facilitated through a process of capture from free flow onto the luminal surface of activated endothelial cells, followed by their adhesion and transendothelial migration (transmigration) into the underlying affected tissue. However, the mechanisms that support the preferential and context-dependent recruitment of monocyte subpopulations are still not fully understood. Therefore, we have developed a method that allows the recruitment of different monocyte subpopulations to be simultaneously visualized and measured under flow. This method, based on time-lapse confocal imaging, allows for the unambiguous distinction between adherent and transmigrated monocytes. Here, we describe how this method can be used to simultaneously study the recruitment cascade of pro-angiogenic and non-angiogenic monocytes in vitro. Furthermore, this method can be extended to study the different steps of recruitment of up to three monocyte populations.

Here, we present an integrated protocol that measures monocyte subpopulation trafficking under flow in vitro by use of specific surface markers and confocal fluorescence microscopy. This protocol can be used to explore sequential recruitment steps as well as to profile other leukocyte subtypes using other specific surface markers.

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development ...

Time-lapse Imaging of Mouse Macrophage Chemotaxis

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a chemical. Chemokinesis and chemotaxis are fundamental for the mobilization and deployment of immune cells. For example, chemokines (chemotactic cytokines) can rapidly recruit circulating neutrophils and monocytes to extravascular sites of inflammation. Chemoattractant receptors belong to the large family of G protein-coupled receptors. How chemoattractant (i.e., ligand) gradients direct cell migration via G protein-coupled receptor signaling is not yet fully understood. In the field of immunology, neutrophils are popular model cells for studying chemotaxis in vitro. Here we describe a real-time two-dimensional (2D) chemotaxis assay tailored for mouse resident macrophages, which have traditionally been more difficult to study. Macrophages move at a slow pace of ~1 &#181;m/min on a 2D surface and are less well suited for point-source migration assays (e.g., migration towards the tip of a micropipette filled with chemoattractant) than neutrophils or Dictyostelium discoideum, which move an order of magnitude faster. Widely used Transwell assays are useful for studying the chemotactic activity of different substances, but do not provide information on cell morphology, velocity, or chemotactic navigation. Here we describe a time-lapse microscopy-based macrophage chemotaxis assay that allows quantification of cell velocity and chemotactic efficiency and provides a platform to delineate the transducers, signal pathways, and effectors of chemotaxis.

Here we describe methods using time-lapse, phase-contrast microscopy to image mouse resident peritoneal macrophages in a chemotactic complement C5a gradient. The protocols can be extended to other immune cells.

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a ...

Prediction of Red Blood Cell Antibody Significance Using the Monocyte-Macrophage Assay

Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, and foreign pathogens. The phagocytic capacity of monocytes versus macrophages is a concept that is not well understood. Here, we aim to examine a difference in the phagocytosis of monocytes versus macrophages, specifically M1/M2 macrophages, against various opsonized red cells using a modified and updated version of the established monocyte monolayer assay (MMA). Peripheral blood mononuclear cells (PBMCs) were isolated from donor buffy coats. Using purified monocytes, inflammatory M1 and anti-inflammatory M2 macrophages were produced by in vitro culture and polarization. M1/M2 cells were harvested and used in an MMA-like assay, which we refer to as the M-MA, to decipher clinically significant phagocytosis of various red cell antibodies. A phagocytic index (PI) &#62; 5 was deemed clinically significant phagocytosis with the use of monocytes. A phagocytic index (PI) &#62; 12 was deemed clinically significant phagocytosis with the use of M1/M2 macrophages. M2 macrophages demonstrate an increased ability to phagocytose opsonized RBCs compared to monocytes and M1s. The same weak antibody (anti-S) yields significant phagocytosis with only M2 macrophages (PI=43) but not M1s (PI=2) or monocytes (PI=0), and this was demonstrated repeatedly using various antibodies. The use of M2 macrophages instead of monocytes may allow for more accurate results as these cells are more phagocytic, offering further clinical relevance to the assay. Further studies with different antibodies to red blood cells, including validation of the monocyte-macrophage assay (M-MA) with antibodies having known clinical significance, may show the M-MA&#160;more useful to help predict clinically significant red cell alloantibodies and transfusion reactions. This method will advance the field of transfusion medicine and immunology.

We have developed enhancements and updated methods for the existing monocyte-monolayer assay (MMA), in which macrophages are used to help better predict the clinical relevance of red cell alloantibodies in transfusion medicine and immunology. This assay is named the monocyte-macrophage assay (M-MA).

Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, ...

Analyzing Ex Vivo Metabolic Flux in Splenic and Cardiac Macrophages and Bone Marrow Monocytes

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, pro-inflammatory (i.e., M1-like) monocytes/macrophages display more reliance on anaerobic glycolysis and less reliance on mitochondrial oxidative phosphorylation, whereas anti-inflammatory (M2-like) macrophages display more reliance on glucose and fatty acid oxidation in the mitochondria. Here, we describe in-depth protocols for extracting macrophages from the two major monocyte/macrophage reservoirs in the body, the spleen and bone marrow, as well as injured tissues such as the heart following myocardial infarction.
Macrophages or monocytes are extracted by immunomagnetic sorting by using antibody-tagged microbeads, which easily bind to cells without compromising their phenotypes. The extracted cells are then cultured in 96-well plates, followed by extracellular flux analysis using a metabolic flux analyzer. Both glycolysis and mitochondrial oxidative phosphorylation can be measured simultaneously in small numbers of cells (as little as 2-3 &#215; 105 cells). This method can easily be performed in 1 day and produces reliable and repeatable results. Ultimately, these methods help to enhance our understanding of metabolic changes during immune and inflammatory responses to injury and disease, which could lead to the development of novel therapeutic targets for immunometabolic pathways.

Here, we describe in detail methods for extracting macrophages from the bone marrow, spleen, and infarcted heart, and subsequently assessing metabolic flux in live cells.

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, ...

Reprograming Model of Human Monocyte-derived Macrophages for In-vitro Assays

Cells of the monocyte-macrophage lineage are multifunctional and found in almost all body tissues. They coordinate innate and adaptive immunity&#180;s initiation and resolution phases, significantly affecting protective immunity and immune-mediated pathological injury. While tissue-resident macrophages are key players in maintaining homeostasis in a steady state, large amounts of monocytes are recruited from the peripheral blood into the tissue following damage or inflammatory insults. Monocyte-derived macrophages (M-DM) can differentiate into many dynamic subtypes, and their phenotypes and functions depend on the local tissue environment. 
To compare different stimuli or environmental conditions during M-DM differentiation and polarization, we standardized an in-vitro model of human nonpolarized M-DM M0 and some cardinal cytokine-polarized macrophages, IFN&#947;/LPS-derived M1, IL-4-derived M2a, and IL-10 or dexamethasone-derived M2c, to analyze skewing reprogramming of M-DM by flow cytometry and real-time PCR. We found that CD64, CD206, CD163, CD14, and MERTK can clearly discriminate unpolarized M0 and polarized M1, M2a, and M2c by flow cytometry. Moreover, we defined IRF1 and CXCL10 as specific genes for classical IFN&#947;/LPS-derived M1-, IRF4-, CCL22-, and TGM2-specific transcripts for IL-4-derived M2a, and the MERTK gene for dexamethasone-derived M2c. To summarize, our standardized M-DM protocol could give the cardinal in-vitro map to analyze the differentiation and polarization of human M-DM under diverse stimuli.

Monocytes and macrophages are very plastic and reprogrammable immune cells crucial for health and disease. They sense and respond to a broad range of stimuli by adopting specific differentiation programs and phenotypes. We have standardized an in-vitro model to study macrophage polarization and reprogramming, providing a valuable tool for research.

Cells of the monocyte-macrophage lineage are multifunctional and found in almost all body tissues. They coordinate innate and adaptive immunity&#180;s ...

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

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