This work was supported by the NSF Major Research Instrument mechanism under grant numbers 1626093 and 1919583.

NOTE: All medium preparation and cell culture techniques are carried out under aseptic conditions using a biosafety cabinet with laminar flow. All culture incubation steps described are carried out using an incubator designed to maintain an atmosphere of 37 &#176;C, 5% CO2, and 95% humidity.
1. Cell culture
<ol>
	<li>Preparation of growth medium
	<ol>
		<li>For MSC and LADMAC, add 50 mL of FBS and 5 mL of 100x antibiotic/antimycotic mix to 500 mL of high glucose DMEM.</li>
		<li>For the M&#934;, add 50 mL of FBS, 100 mL of LADMAC condition medium (prepared following the steps of section 1.2), and 5 mL of 100x antibiotic mix to 500 mL of high glucose DMEM. 
		&#8203;NOTE: LADMAC cells produce colony-stimulating factor (CSF-1) necessary to support the growth of the M&#934; cells.</li>
	</ol>
	</li>
	<li>Preparation of LADMAC conditioned medium
	<ol>
		<li>To seed LADMAC cells from frozen stock, add 19 mL of growth medium from section 1.1 in each of the five T75 cm2 cell-culture treated flasks and place in the cell culture incubator for 15 min to equilibrate to 37 &#176;C.</li>
		<li>Meanwhile, quick thaw one aliquot of 106 frozen cells by incubating in a 37 &#176;C water bath for 2-3 min or until it gets thawed. Add the cells to 9 mL of fresh MSC growth medium in a 15 mL or 50 mL sterile conical tube and centrifuge at 125 x g in a swinging bucket rotor for 5 min.</li>
		<li>Aspirate or decant the supernatant, add 5 mL of fresh growth medium, and gently pipette up and down to resuspend the cell pellet.</li>
		<li>Add 1 mL of the cell suspension to each of the five T75 cm2 flasks using a sterile serological pipette and place them&#160;in the cell culture incubator.</li>
		<li>Every 2-3 days, add an additional 10 mL of medium over a 7-10-day period. Then, collect the cells and the supernatant into sterile 50 mL conical tubes using a sterile serological pipette and centrifuge at 125 x g for 5 min.</li>
		<li>Separate the supernatant from the cell pellet and sterile filter the supernatant using a 0.2 &#181;m sterile single-use vacuum filter unit.
		<ol>
			<li>Remove the aspirator assembly from the package and connect using vacuum tubing to the aspirator pump. Pour the supernatant into the top compartment and replace the cover. Turn on the aspirator pump to filter into the bottom compartment.</li>
			<li>Prepare aliquots by pipetting into 50 mL sterile conical tubes and store at -20 &#176;C.</li>
		</ol>
		</li>
		<li>To freeze the cell pellet, resuspend in freezing medium at 106 cell/mL, place in a freezing container, and then place them in a -80 &#176;C freezer. After 24 h, transfer to LN2 storage.</li>
	</ol>
	</li>
	<li>Propagate cells in preparation for co-culture
	<ol>
		<li>To seed MSC and M&#934; cells from the frozen stock, equilibrate 9 mL of appropriate growth medium prepared in section 1.1 in each of the four 100 mm cell culture treated dishes for each cell type and place in the cell culture incubator for 15 min.</li>
		<li>Meanwhile, quick thaw the aliquots of 106 frozen cells by incubating them in a 37 &#176;C water bath for 2-3 min or until just thawed. Then, add the thawed MSC cells to 9 mL of fresh MSC growth medium and add the thawed M&#934; cells to 9 mL fresh M&#934; growth medium in 15 or 50 mL sterile conical tubes and centrifuge at 125 x g in swinging bucket rotor for 5 min.</li>
		<li>Aspirate or decant the supernatant and resuspend each pellet in 4 mL of the appropriate fresh growth medium by gently pipetting up and down. Add 1 mL of the cell suspension to each of the four 100 mm dishes for each cell type that have been equilibrated in step 1.3.1.</li>
		<li>Return the dishes with cells to the incubator. Change the medium every 2-3 days until 70%-80% confluent.</li>
	</ol>
	</li>
</ol>
2. Seed MSC in experimental dishes, Day 1
NOTE: Prior to seeding the MSC, design the experimental 96-well plate layout for fluorescent spectrophotometry and the chamber slide for dynamic imaging. For the 96-well plate, flank experimental wells with wells containing cells but do not use them in the assay. Mark at least four wells to be used for the reagent blank (RBL). See Table 1 for an example 96-well template and Table 2 for an example of a 4-well chamber slide template. Black or white 96-well plates with clear bottoms are recommended. A 1.5 mm borosilicate glass chamber slide is optimal, but the number of chambers can be adjusted dependent on the study design. A summary flow chart of the methods that follow is included in Figure 2.
<ol>
	<li>At 70%-80% confluency, aspirate the growth medium from the cultures in 100 mm dishes and add 5 mL of PBS to isolate the MSC.</li>
	<li>Aspirate the PBS, add 2 mL of 0.05% trypsin/EDTA, and place it in the culture incubator for 3-5 min.</li>
	<li>After 3 min, check the cell detachment using an inverted microscope. If the cells are detached, continue to the next step; if not, continue incubation for another 1-2 min. Do not incubate longer than 5-6 min.</li>
	<li>Add 5 mL of fresh growth medium to the dish with the detached cells. Rinse the dish using the trypsin/medium mixture and collect cells into a clean, sterile 50 mL conical tube using a sterile serological pipette.</li>
	<li>Centrifuge for 5 min at 125 x g. Aspirate the supernatant and resuspend the cell pellet in 10 mL of fresh growth medium by gently pipetting up and down.</li>
	<li>To count the cells, add 10 &#181;L of the cell suspension to 30 &#181;L of 0.4% trypan blue solution in a 1.5 mL microcentrifuge tube. Then, pipette 10 &#181;L of the mixture under the coverslip of a hemocytometer counting chamber.</li>
	<li>Using an inverted or bright field microscope, with the 10x objective, count the unstained (viable) cells in four of the 1 mm2 squares. Calculate the cell number using the formula: cells/mL = cell count/# 1 mm2 squares x dilution factor x 104. 
	NOTE: For this example, the number of 1 mm2 squares counted is four, and the dilution factor is 4.</li>
	<li>Use the equation C1V1 = C2V2 to calculate for and prepare a 1 x 105 cell/mL suspension. Prepare 1 mL of cell/mL suspension for every column of the 96-well plate to be filled and 0.75 mL for every chamber of the 4-well chamber slide to be filled according to the plate design. 
	NOTE: C1 = cell count, V1 = what is being calculated, C2 = 1 x 105, V2 = 5.50 mL.</li>
	<li>Determine V1, subtract it from the desired 5.50 mL of the total volume to determine the volume of the fresh medium needed to prepare the suspension. Add the volume of cells (V1) from the original suspension to the volume of fresh medium for a total of 5.50 mL with 1 x 105 cells/mL.</li>
	<li>Add 100 &#181;L of 1 x 105 cell/mL suspension to each well of the 96-well plate according to the plate design using a multichannel micropipettor and 530 &#181;L to each of the wells of the chamber slide using a micropipette according to the plate design. Incubate overnight. 
	NOTE: For this experiment, MSC&#160;are plated in columns 1, 2, 3, and 6 of the 96-well plate (Table 1) and wells 1 and 2 of the 4-well chamber slide (Table 2). Therefore, at least 5.50 mL of a 1 x 105 cell/mL suspension is prepared. M&#934; at 80% confluence are treated with inflammatory mediators the same day the MSC&#160;are seeded in the experimental plates.</li>
</ol>
3. Activate the M&#934; with IFN-&#947;, Day 1
<ol>
	<li>Prepare the activation medium and IFN-&#947; stock.
	<ol>
		<li>For 200 mL of activation medium, add 40 mg of BSA to 200 mL of serum-free high glucose DMEM and sterile filter using a 0.2 &#181;m sterile single-use vacuum filter unit.</li>
		<li>To prepare 0.1% BSA/PBS, add 20 mg of BSA to 20 mL of PBS. Vortex to dissolve. Use a 0.2 &#181;m sterile syringe filter to filter into a clean, sterile 50 mL conical tube.</li>
		<li>Add 1 mL of the sterile 0.1% BSA/PBS solution to 100 &#181;g of lyophilized IFN-&#947; to achieve a 100 &#181;g/mL of stock solution. Store in 50 &#181;L aliquots at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Activate the M&#934; with IFN-&#947;
	<ol>
		<li>Add 2.5 &#181;L of 100 &#181;g/mL of IFN-&#947; stock for every 1 mL of the medium needed. For every 100 mm dish to be activated, prepare 10 mL of activation medium containing IFN-&#947; at a concentration of 250 ng/mL.</li>
		<li>Aspirate the growth medium from the M&#934; cultures and replace it with 5 mL of the activation medium without IFN- &#947; to rinse.</li>
		<li>Aspirate the medium used for rinsing and replace it with 10 mL of IFN- &#947; supplemented activation medium for the experimental dish and with 10 mL of non-supplemented activation medium for the control. Incubate the cultures for 16-24 h.</li>
	</ol>
	</li>
</ol>
4. Isolate the M&#934; and prepare the co-cultures, Day 2
<ol>
	<li>Remove the activation medium from the M&#934; cells and replace it with 5 mL of fresh growth medium. Scrape gently with a cell lifter and collect the cells into a 50 mL conical tube.</li>
	<li>Count the cells using trypan blue exclusion and hemocytometry as described for MSC in, steps 2.6-2.7.</li>
	<li>Using the equation in steps 2.8-2.9, prepare two separate suspensions of 2 x 105 cells/mL, one with cells from the control and one with cells from the treated M&#934; cultures. 
	NOTE: Prepare 1 mL of cell/mL suspension for every column of the 96-well plate to be filled and 0.75 mL for every chamber of the 4-well chamber slide to be filled according to the plate design.</li>
	<li>Gently aspirate the medium from the experimental wells of the 96-well plate containing MSC. Using a multichannel micropipette, add 100 &#181;L of the treated and the control M&#934; cell suspensions in the appropriate experimental wells with and without MSC according to the plate design. Incubate overnight.</li>
	<li>Gently aspirate the medium from the 4-well chamber slide containing MSC, and, using a micropipette, add 530 &#181;L of M&#934; cell suspension to the appropriate wells according to the design. Incubate overnight.</li>
</ol>
5. Phagocytosis assay, kinetic fluorescent 96-well plate read, Day 3
<ol>
	<li>Set the assay parameters
	<ol>
		<li>On the day of the assay, turn on the fluorescent plate reader and computer-set the temperature on the system to 37 &#176;C.</li>
		<li>Open the system Pro software and check the instrument icon in the upper-left corner to determine whether the instrument is connected to the computer. If the red circle with a line is visible, click on the instrument icon and choose the instrument in the pop-up window to make the connection.</li>
		<li>Select New Experiment to access the Plate Set-Up Helper pop-up window. From the Plate Set-Up Helper menu, select Configure Your Acquisition Settings.</li>
		<li>Select Monochromator from the settings menu for the optical configuration. Select FL (fluorescence) for the read mode and Kinetic for the read type.</li>
		<li>In the same configurations window, under Category, click on Wavelengths, and then set bandwidths to 9 nm for excitation and 15 nm for emission.</li>
		<li>Set the Number of Wavelength Pairs to 1. Set the wavelengths LM1 to 510 nm for excitation and to 540 nm for emission.</li>
		<li>Continue through the categories and select Plate Type next. Select the option that matches the plate type being used. 
		NOTE: It is important that the selection matches the plate type. The read heights are set by the system according to the selection.</li>
		<li>Next, select Read Area and highlight the area of the 96-well plate included in the experimental design.</li>
		<li>Select PMT and Optics, set PMT Gain to High and Flashes per read to 6. Check the box next to Read from Bottom if using a clear-bottom plate.</li>
		<li>Select Timing and set for 10 min intervals over a 70 min period.</li>
		<li>Select Shake, check the Before First Read box, and set for 5 s. Check the Between Reads box and set for 3 s. Set shake intensity for both to Low and Linear.</li>
		<li>Close the window. When the Plate Set-Up Helper window pops up, select Configure Your Plate Layout. Highlight the BL wells of the plate design and click on Plate Blank.</li>
		<li>Highlight each of the experimental rows, click on Add, name the group, and then select a color.</li>
		<li>Under Assignment Options below the plate design, select Series, and then define the series in the window and click on OK. Repeat for each group.</li>
	</ol>
	</li>
	<li>Prepare the zymosan suspension
	<ol>
		<li>While waiting for the system temperature to reach 37 &#176;C, resuspend 1 mg of zymosan particles in 5 mL of the live-cell imaging medium.
		<ol>
			<li>Add 1 mL of live-cell imaging medium to the vial containing the particles and collect them into a glass culture tube. Rinse the vial with an additional 1 mL of imaging solution and transfer to the same glass tube to ensure that all the particles are transferred. Add 3 mL of additional imaging solution to achieve a 0.2 mg/mL of particle suspension.</li>
		</ol>
		</li>
		<li>Vortex with quick pulses for 30-60 s. Then, use a probe sonicator to sonicate with 60 quick pulses. 
		NOTE: It is very important to create a homogenous suspension of particles and not let the suspension sit too long before adding to the experimental wells. Clumping recurs rapidly. The particle per cell density may need to be optimized depending on the source, treatment, and density of M&#934; used. In the studies presented, conjugated zymosan particles were used. However, conjugated E. coli and S. aureus are also available.</li>
	</ol>
	</li>
	<li>Add zymosan and read the plate
	<ol>
		<li>Aspirate the medium from the experimental wells and rinse 1x with 100 &#181;L of the live-cell imaging solution. Rinse the RBL wells with the live-cell imaging solution as well.</li>
		<li>Aspirate the live-cell imaging solution from experimental and RBL wells and replace it with 100 &#181;L of the zymosan particle suspension prepared in section 5.2.</li>
		<li>Open the plate tray of the fluorescent reader using the touchpad interface. Set the plate, without the lid, in the tray with the A1 well in the upper-left corner. Close the tray using the touchpad and click on the green Read button in the top menu.</li>
		<li>When the read is complete, save the file to the appropriate folder and export the data in a spreadsheet format by clicking on File and selecting Export.</li>
		<li>Calculate the mean &#177; SEM relative fluorescence for each replicate group at each time point (10 min, 20 min, 30 min, etc.) in the spreadsheet (Supplementary File 1) and transfer the data to graphing software.</li>
		<li>Use a line graph format to present the data and apply two-way ANOVA (Time x Treatment/MSC as factors) followed by Tukey&#39;s multiple comparisons test to determine differences between individual groups. 
		&#8203;NOTE: While the 96-well plate read is in progress, prepare the dynamic imaging plate for time-lapse image acquisition. Ensure that the dynamic imaging proceeds with the analysis of one experimental well at a time.</li>
	</ol>
	</li>
</ol>
6. Phagocytosis assay, dynamic imaging, Day 3
<ol>
	<li>Turn on the imaging system and computer. Double click to open the software. Select the Pro program mode.</li>
	<li>Check the stage area to ensure no samples are present and nothing is impeding the movement of the stage. Then, click on Calibrate Now.</li>
	<li>Under the Incubation menu on the sidebar on the right, check the box next to H Unit XL and set it to 37 &#176;C. 
	NOTE: Wait until the incubated stage is almost to the temperature before preparing the samples for imaging.</li>
	<li>Rinse the first experimental well with 750 &#181;L of imaging medium and replace it with 400 &#181;L of the zymosan particle suspension prepared in section 5.2. Leave the remaining wells in the growth medium. 
	NOTE: It may be necessary to vortex and sonicate the particle suspension again briefly if it has settled for more than 15 min.</li>
	<li>Place the slide on the incubated stage of the imaging system. Set a timer for 10 min.</li>
	<li>Use the imaging software, select Brightfield under the Locate tab, and then click on the eyepiece icon in the system configuration window below. With the 10x objective in place, use the eyepiece and focusing knob to focus on the cells.</li>
	<li>Select the Acquisition tab, use the Experiment drop-down menu, and then select a set of wavelengths that includes EGFP filter set to accommodate 509 nm excitation 533 nm emission wavelengths of the pH-sensitive dye.</li>
	<li>When there is ~2 min left on the timer, use the software to change the objective to 20x by clicking on the 20x icon in the objective menu.
	<ol>
		<li>Click on the Channels menu, select EGFP filters and use the Exposure menu to set the exposure time to 400 ms to detect the pH-sensitive green fluorophore once it has been enclosed in the phagolysosome.</li>
		<li>Click on Live and adjust the focus using the focusing knob.</li>
	</ol>
	</li>
	<li>Click on Stop&#160;to turn off the light and check the box next to the Time-lapse experimental setting at the top.</li>
	<li>In the Focusing Strategy menu select Software Autofocus. From the drop-down in the Autofocus menu, select Smart and Coarse settings to reduce the time of exposure to light while the autofocus is running.</li>
	<li>In the Time-Lapse menu, set the desired number of acquisitions to 30-60 and the interval time to 1 min.</li>
	<li>After the time-lapse parameters are set and after 10 min of addition of particles to the first well, click on the Start Experiment button to begin acquisition.</li>
	<li>When the acquisition is complete using the first experimental well, repeat steps 6.4-6.12 for each of the remaining experimental wells.</li>
	<li>Save all the experiment files with the date and parameters in the title to the appropriate folder.</li>
	<li>Close the software. Reopen the software in Processing mode and export the files in the MP4 format using the Export menu.</li>
</ol>

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V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+transfer+via+tunneling+nanotubes+is+an+important+mechanism+by+which+mesenchymal+stem+cells+enhance+macrophage+phagocytosis+in+the+in+vitro+and+in+vivo+models+of+ARDS.">Mitochondrial transfer via tunneling nanotubes is an important mechanism by which mesenchymal stem cells enhance macrophage phagocytosis in the in vitro and in vivo models of ARDS.</a> Stem Cells. 34 (8), 2210-2223 (2016).</li><li>Chaplin, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+the+immune+response.">Overview of the immune response.</a> The Journal of Allergy and Clinical Immunology. 125 (2), 3-23 (2010).</li><li>Lim, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+the+mechanisms+of+nonopsonic+uptake+of+cryptococci+by+macrophages.">Characterizing the mechanisms of nonopsonic uptake of cryptococci by macrophages.</a> Journal of Immunology. 200 (10), 3539-3546 (2018).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-&#947;+inhibits+nonopsonized+phagocytosis+of+macrophages+via+an+mTORC1-c/EBP+pathway.">Interferon-&#947; inhibits nonopsonized phagocytosis of macrophages via an mTORC1-c/EBP pathway.</a> Journal of Innate Immunity. 7 (2), 165-176 (2015).</li><li>Lindner, B., Burkard, T., Schuler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+assays+with+different+PH-sensitive+fluorescent+particles+and+various+readouts.">Phagocytosis assays with different PH-sensitive fluorescent particles and various readouts.</a> Biotechniques. 68, 245-250 (2020).</li><li>Kapellos, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+real+time+imaging+platform+to+quantify+macrophage+pahgocytosis.">A novel real time imaging platform to quantify macrophage pahgocytosis.</a> Biochemical Pharmacology. 116, 107-119 (2016).</li><li>Takahashi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+quantitation+of+platelet+phagocytosis+by+monocytes+using+a+pH-sensitive+dye,+pHrodo-SE.">Flow cytometric quantitation of platelet phagocytosis by monocytes using a pH-sensitive dye, pHrodo-SE.</a> Journal of Immunological Methods. 447, 57-64 (2017).</li></ol>

The authors have no conflicts of interest to declare.

Analysis of phagocytosis using bioparticles conjugated to a pH-sensitive dye is a relatively new tool that has proven advantageous over traditional fluorescently labeled particles12,19,20. With traditional fluorescent-labeled particles, only end-point analysis is feasible. Detection is carried out with fluorescent microscopy and/or spectrofluorometry after washing or quenching particles that have not been taken up by the phagocyte. Quantitative data derived from spectrofluorometry has the potential to detect non-engulfed particles, and image analysis to quantify only intracellular particles is tedious and time-consuming using traditional fluorescent microscopy systems14. Bioparticles conjugated to pH-sensitive dyes fluoresce only in an acidic environment such as the phagolysosome, and, therefore, the tedious washing and quenching steps are unnecessary14. Additionally, pH-sensitive labeled bioparticles provide the advantage of providing kinetic data that would not be acquired easily with FITC or other fluorophore-conjugated bioparticles.
Studies using this new tool have employed flow cytometry and/or imaging platforms to generate a quantitative and kinetic measurement of phagocyte activity19,20,21. Flow cytometry is advantageous if there is a limited phagocyte population or if one is interested in sorting for downstream analyses such as genetic screens19. MSC&#160;are known to regulate the M&#934; phenotype through both direct contact and through soluble factors. Preliminary studies have shown that conditioned medium from MSC suppressed M&#934; phagocytosis, and therefore, this protocol was designed to determine the changes in M&#934; phagocytic activity while in direct co-culture with MSC. Under these conditions, spectrofluorometry to quantify and dynamic imaging to visualize and confirm phagocytic activity are most appropriate.
Critical to the success of these methods is the optimization of cell densities. The MSC&#160;need to be plated at a density that allows for optimal cell contact with the M&#934; cells. M&#934; need to be seeded in mono and co-cultures at a density that not only allows for optimal contact but also allows for optimal fluorescence detection. Too low of a density will result in low incorporation into the phagolysosome and small or flat changes in relative fluorescent units (Figure 3B). If M&#934; are seeded at too high of a density, phagocytosis increases rapidly, and detection of differences between groups is masked (Figure 3C). Optimizing the concentration of pH-sensitive dye labeled zymosan particles per cell number is also critical. Preliminary experiments should be performed to optimize cell densities of MSC and M&#934;, and the number of particles per M&#934; cell. Additionally, these optimization steps should be taken if other labeled bioparticles are used, such as E. coli or S. aureus.
The appropriate imaging medium is also critical. The medium for both methods should be a buffered medium and should not contain phenol red. Phenol red will mute the detection of the fluorescent emission. Also, any additive or condition that can acidify the medium will prematurely activate the labeled particles and introduce elevated background. The reagent blank is critical for identifying the potential acidification of the medium.
Not only can these protocols be used to investigate MSC regulation of M&#934; phagocytosis of a variety of pH-sensitive bioparticles, but the method can also be applied to a variety of co-culture models that include neutrophils and other phagocytes. Additionally, by using this model, molecules and pathways suspected of being involved in cell contact-mediated regulation of macrophage phagocytic activity can be probed via siRNA or CRISPR mediated down-regulation to validate or refute suspected molecular targets. Potential targets include adhesion molecules, phagocytic receptors, and integrin molecules.
Work using these methods will uncover new information regarding the signaling mechanisms underlying the increased phagocytic responses of M&#934; following cell contact interactions with MSC, thereby furthering our understanding of the role MSC play in regulating innate immunity. Studies such as these address an understudied area and will yield a complete picture of the influence MSC have on macrophage activity, which is necessary for a full understanding of their role in immunity.

Mesenchymal stem cells (MSC) are progenitor cells that give rise to connective tissue cells. MSC are present in adult mammalian tissues and can be isolated from the bone marrow1. Due to their immunomodulatory properties, these cells are widely studied2. Early studies focused on MSC regulation of T-cells3,4,5,6 but more recently, their regulation of macrophage cells (M&#934;), a major cellular component of innate immunity, has received increased attention7,8,9,10,11,12,13,14. The importance of MSC-M&#934; interaction in the treatment of inflammatory disease is underscored by the fact that depletion of monocytes/macrophages abrogates the therapeutic effects of MSC in animal models8. Here, the focus is the cell contact interaction of the MSC with M&#934;. MSC&#160;have the capacity to&#160;regulate the phenotype of M&#934; by promoting the switch from inflammatory to anti-inflammatory responses, leading to tissue repair activities8,9,10,11, and much has been done to demonstrate these regulatory mechanisms. Under other circumstances, MSC can support or exacerbate a&#160;M&#934;-driven inflammatory response12,13 and enhance M&#934; phagocytic activity14,15. However, there is a critical lack of existing data that identifies the mechanisms whereby and the conditions under which MSC regulate M&#934; phagocytic activity.
M&#934; have families of receptors that recognize either opsonized (antibody or complement coated) or non-opsonized pathogens leading to phagocytosis16. The activation and activity of the latter is less well studied17. In a non-inflammatory in vitro environment, MSC&#160;enhance M&#934; phagocytosis of non-opsonized pathogens13. However, recognition of non-opsonized pathogens by M&#934; may be reduced after exposure to an inflammatory environment produced by lymphocytes during an adaptive immune response. IFN-&#947;, released by natural killer cells and effector lymphocytes, has an inhibitory effect on M&#934; phagocytosis of non-opsonized particles18. A co-culture model was developed to study the mechanisms of MSC direct contact regulation of M&#934; phagocytosis. The goal of the experiment presented here is to determine whether MSC&#160;regulate M&#934; phagocytosis of non-opsonized pathogens after M&#934; have been exposed to IFN-&#947; (Figure 1).

<table><tbody><tr><td>96 Well Black Polystyrene Microplate</td><td>MilliporeSigma</td><td>CLS3603-48EA</td><td></td></tr><tr><td>0.4% trypan blue solution</td><td>MilliporeSigma</td><td>T8154-20ML</td><td></td></tr><tr><td>15 mL and 50 mL Conical Sterile Polypropylene Centrifuge Tubes</td><td>ThermoFisher</td><td>339653</td><td></td></tr><tr><td>4-well Chambered Coverglass w/ non-removable wells</td><td>ThermoFisher</td><td>155382PK</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100X)&amp;nbsp;Gibco</td><td>ThermoFisher</td><td>15240096</td><td></td></tr><tr><td>Axiobserver 7 Imaging System</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>MilliporeSigma</td><td>A8806-1G</td><td></td></tr><tr><td>Cell lifter</td><td>MilliporeSigma</td><td>CLS3008-100EA</td><td></td></tr><tr><td>Culture flasks, tissue culture treated, surface area 75&amp;nbsp;cm&lt;sup&gt;2&lt;/sup&gt;, canted neck, with cap, filtered</td><td>MilliporeSigma</td><td>C7231-120EA</td><td></td></tr><tr><td>D1 ORL UVA [D1]</td><td>ATCC</td><td>CRL-12424</td><td>Mouse MSC Cell Line</td></tr><tr><td>DMEM, High Glucose</td><td>ThermoFisher</td><td>11965092</td><td></td></tr><tr><td>Fetal Bovine Serum, qualified, heat inactivated</td><td>ThermoFisher</td><td>16140071</td><td></td></tr><tr><td>Hemocytometer</td><td>FisherScientific</td><td>02-671-51B</td><td></td></tr><tr><td>I-11.15</td><td>ATCC</td><td>CRL-2470</td><td>Mouse M&amp;Phi; Cell Line</td></tr><tr><td>LADMAC Cell Line</td><td>ATCC</td><td>CRL-2420</td><td>LADMAC cells secrete the growth factor colony stimulating factor 1 (CSF-1).</td></tr><tr><td>Live-Cell Imaging solution</td><td>ThermoFisher</td><td>A14291DJ</td><td></td></tr><tr><td>PBS, pH 7.4</td><td>ThermoFisher</td><td>10010031</td><td></td></tr><tr><td>pHrodo Green Zymosan Bioparticles Conjugate</td><td>ThermoFisher</td><td>P35365</td><td></td></tr><tr><td>Recombinant Murine IFN-&amp;gamma;</td><td>Preprotech</td><td>315-05</td><td></td></tr><tr><td>Spectramax i3X</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Sterile Single Use Vacuum Filter Units, 250 mL, 0.2 &amp;micro;m</td><td>ThermoFisher</td><td>568-0020</td><td></td></tr><tr><td>Sterile syringe filters, 0.2 micrometer</td><td>ThermoFisher</td><td>723-2520</td><td></td></tr><tr><td>Tissue-culture treated culture dishes, 100 mm x 20 mm</td><td>MilliporeSigma</td><td>CLS430167-100EA</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>ThermoFisher</td><td>25300054</td><td></td></tr></tbody></table>

mesenchymal stem cell regulation of macrophage phagocytosis; quantitation and imaging

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics have been at the forefront. They interact with and regulate immune cell activity. The focus of this study is the MSC regulation of macrophage phagocytic activity. Macrophage (M&#934;) phagocytosis is an important part of the innate immune system response to infection, and the mechanisms through which MSC modulate this response are under active investigation. Presented here is a method to study M&#934; phagocytosis of non-opsonized zymosan particles conjugated to a pH-sensitive fluorescent molecule while in co-culture with MSC. As phagocytic activity increases and the labeled zymosan particles are enclosed within the acidic environment of the phagolysosome, the fluorescence intensity of the pH-sensitive molecule increases. With the appropriate excitation and emission wavelengths, phagocytic activity is measured using a fluorescent spectrophotometer and kinetic data is presented as changes in relative fluorescent units over a 70 min period. To support this quantitative data, the change in the phagocytic activity is visualized using dynamic imaging. Results using this method demonstrate that when in co-culture, MSC enhance M&#934; phagocytosis of non-opsonized zymosan of both naive and IFN-&#947; treated M&#934;. These data add to the current knowledge of MSC regulation of the innate immune system. This method can be applied in future investigations to fully delineate the underlying cellular and molecular mechanisms.

After calculating mean &#177; SEM for each group at all time points, the data is presented in line graph format with the Y-axis as the Relative Fluorescent Intensity and the X-axis as Time. Supplementary File 1 provides an example of raw data from a kinetic read of the 96-well plate in a spreadsheet format.
In this study, the optimal results presented in Figure 3A, and Table 3 demonstrate that 1) co-culture with MSC&#160;enhances the phagocytic activity of the macrophage, 2) IFN-y&#160;treatment reduces the activity of the macrophage, and 3) co-culture with MSC partially rescues M&#934; phagocytic activity. Optimal cell densities are critical in these studies, and when M&#934; are plated at too low of a density, changes in fluorescence intensity cannot be detected (Figure 3B). Figure 3C represents data from a study where the M&#934; were plated at too high of a density and fluorescence intensity is elevated rapidly in all groups, and differences cannot be discerned. The dynamic imaging videos confirm that the fluorescent intensity changes result from phagocytosis and not acidification of the medium. They also provide qualitative data and a visual representation of the rate and extent of phagocytic activity (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62729/62729fig01.jpg" /> 
Figure 1: An illustration depicting the central question of the data presented, which is &#34;Can MSC recover the phagocytic activity of IFN-&#947; treated M&#934;?&#34;. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62729/62729fig01large.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/62729/62729fig2v2.jpg" /> 
Figure 2: Overview of the co-culture and phagocytosis assay workflow. An outline of the workflow for quantitative and qualitative analysis of M&#934; phagocytosis activity in co-culture with MSC. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62729/62729fig2v2large.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/62729/62729fig3v2.jpg" /> 
Figure 3: Representative quantitative data from the kinetic readdemonstrating optimal and suboptimal results. In (A), data are representative of an experiment with optimal M&#934; cell density, in (B) data are representative of an experiment with suboptimal too low M&#934; cell density, and in (C), data are representative of an experiment with suboptimal too high M&#934; cell density. The relative fluorescence intensity measured in relative fluorescent units (RFU) is plotted on the Y-axis, while time is plotted on the X-axis. Note the differences in the range of RFU among optimal and sub-optimal experiments. In A, MSC&#160;partially rescue M&#934; phagocytic activity in the setting of IFN-&#947; suppression over a 70 min period. The RFU is presented as mean &#177; the SEM, n = 6. The analysis was performed using Tukey&#39;s multiple comparisons test after a significant two-way ANOVA, Interaction effect P = 0.0001, Time effect P = 0.0001, and Treatment/MSC effect P = 0.0001. Symbols denote results of multiple comparison tests. * = significant difference between MSC/M&#934; + IFN- &#947; vs M&#934; + IFN-&#947;, &#358; = significant difference between M&#934; vs M&#934; + IFN-&#947;, and &#8224; = significant difference between MSC/M&#934; vs M&#934;. See Table 3 for detailed results of the multiple comparison tests. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62729/62729fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 4: Dynamic imaging videos providing visual confirmation of the cell-specific increase in fluorescence from acidic activation of labeled zymosan particles after incorporation into the M&#934; phagolysosome. Time-lapse settings were acquisition every 1 min over a 30 min period using an exposure time of 400 ms and the EGFP filter set. (A) M&#934; in monoculture, (B) M&#934; in co-culture with MSC, (C) M&#934; treated with IFN-&#947; (250 ng/mL) and (D) M&#934; treated with IFN-&#947; (250 ng/mL) in co-culture with MSC. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62729/62729fig04.zip" target="_blank">Please click here to download this File.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>1</td>
			<td>2</td>
			<td>3</td>
			<td>4</td>
			<td>5</td>
			<td>6</td>
			<td>7</td>
			<td>8</td>
			<td>9</td>
			<td>10</td>
			<td>11</td>
			<td>12</td>
		</tr>
		<tr>
			<td>A</td>
			<td>CBL</td>
			<td>MSC/M&#934;</td>
			<td>MSC/M&#934;+</td>
			<td>M&#934;</td>
			<td>M&#934;+</td>
			<td>CBL</td>
			<td>RBL</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B</td>
			<td>CBL</td>
			<td>MSC/M&#934;</td>
			<td>MSC/M&#934;+</td>
			<td>M&#934;</td>
			<td>M&#934;+</td>
			<td>CBL</td>
			<td>RBL</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C</td>
			<td>CBL</td>
			<td>MSC/M&#934;</td>
			<td>MSC/M&#934;+</td>
			<td>M&#934;</td>
			<td>M&#934;+</td>
			<td>CBL</td>
			<td>RBL</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D</td>
			<td>CBL</td>
			<td>MSC/M&#934;</td>
			<td>MSC/M&#934;+</td>
			<td>M&#934;</td>
			<td>M&#934;+</td>
			<td>CBL</td>
			<td>RBL</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E</td>
			<td>CBL</td>
			<td>MSC/M&#934;</td>
			<td>MSC/M&#934;+</td>
			<td>M&#934;</td>
			<td>M&#934;+</td>
			<td>CBL</td>
			<td>RBL</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>F</td>
			<td>CBL</td>
			<td>MSC/M&#934;</td>
			<td>MSC/M&#934;+</td>
			<td>M&#934;</td>
			<td>M&#934;+</td>
			<td>CBL</td>
			<td>RBL</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>G</td>
			<td>CBL</td>
			<td>MSC/M&#934;</td>
			<td>MSC/M&#934;+</td>
			<td>M&#934;</td>
			<td>M&#934;+</td>
			<td>CBL</td>
			<td>RBL</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>H</td>
			<td>CBL</td>
			<td>MSC/M&#934;</td>
			<td>MSC/M&#934;+</td>
			<td>M&#934;</td>
			<td>M&#934;+</td>
			<td>CBL</td>
			<td>RBL</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Example of a 96-well plate design. CBL - Cell blank; MSC - seeded day 1; M&#934;+ treated and M&#934; untreated seeded day 2; RBL reagent blank added on assay day 3.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1</td>
			<td>2</td>
			<td>3</td>
			<td>4</td>
		</tr>
		<tr>
			<td>MSC/M&#934;</td>
			<td>MSC/M&#934;+</td>
			<td>M&#934;</td>
			<td>M&#934;+</td>
		</tr>
	</tbody>
</table>
Table 2: Example of a 4-well chamber slide design. MSC - plated day 1; M&#934;+ treated and M&#934; untreated plated day 2.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tukey&#39;s multiple comparisons test</td>
			<td>Mean Diff.</td>
			<td>95.00% CI of diff.</td>
			<td>Below threshold?</td>
			<td>Summary</td>
			<td>Adjusted P Value</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0 minutes</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MSC/M&#934; vs. M&#934;</td>
			<td>-3871</td>
			<td>-9495 to 1754</td>
			<td>No</td>
			<td>ns</td>
			<td>0.2836</td>
		</tr>
		<tr>
			<td>MSC/M&#934; + IFN-&#947; vs. M&#934; + IFN-&#947;</td>
			<td>720.3</td>
			<td>-4904 to 6345</td>
			<td>No</td>
			<td>ns</td>
			<td>0.9873</td>
		</tr>
		<tr>
			<td>M&#934; vs. M&#934; + IFN-&#947;</td>
			<td>-77.17</td>
			<td>-5702 to 5548</td>
			<td>No</td>
			<td>ns</td>
			<td>&#62;0.9999</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 minutes</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MSC/M&#934; vs. M&#934;</td>
			<td>-3466</td>
			<td>-9091 to 2159</td>
			<td>No</td>
			<td>ns</td>
			<td>0.3817</td>
		</tr>
		<tr>
			<td>MSC/M&#934; + IFN-&#947; vs. M&#934; + IFN-&#947;</td>
			<td>2326</td>
			<td>-3299 to 7950</td>
			<td>No</td>
			<td>ns</td>
			<td>0.7062</td>
		</tr>
		<tr>
			<td>M&#934; vs. M&#934; + IFN-&#947;</td>
			<td>992</td>
			<td>-4633 to 6617</td>
			<td>No</td>
			<td>ns</td>
			<td>0.968</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20 minutes</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MSC/M&#934; vs. M&#934;</td>
			<td>-1311</td>
			<td>-6936 to 4314</td>
			<td>No</td>
			<td>ns</td>
			<td>0.9303</td>
		</tr>
		<tr>
			<td>MSC/M&#934; + IFN-&#947; vs. M&#934; + IFN-&#947;</td>
			<td>3315</td>
			<td>-2310 to 8940</td>
			<td>No</td>
			<td>ns</td>
			<td>0.422</td>
		</tr>
		<tr>
			<td>M&#934; vs. M&#934; + IFN-&#947;</td>
			<td>3146</td>
			<td>-2478 to 8771</td>
			<td>No</td>
			<td>ns</td>
			<td>0.4689</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30 minutes</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MSC/M&#934; vs. M&#934;</td>
			<td>384.8</td>
			<td>-5240 to 6010</td>
			<td>No</td>
			<td>ns</td>
			<td>0.998</td>
		</tr>
		<tr>
			<td>MSC/M&#934; + IFN-&#947; vs. M&#934; + IFN-&#947;</td>
			<td>2313</td>
			<td>-3312 to 7937</td>
			<td>No</td>
			<td>ns</td>
			<td>0.7098</td>
		</tr>
		<tr>
			<td>M&#934; vs. M&#934; + IFN-&#947;</td>
			<td>8726</td>
			<td>3101 to 14350</td>
			<td>Yes</td>
			<td>***</td>
			<td>0.0005</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>40 minutes</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MSC/M&#934; vs. M&#934;</td>
			<td>2247</td>
			<td>-3377 to 7872</td>
			<td>No</td>
			<td>ns</td>
			<td>0.7278</td>
		</tr>
		<tr>
			<td>MSC/M&#934; + IFN-&#947; vs. M&#934; + IFN-&#947;</td>
			<td>4913</td>
			<td>-712.2 to 10537</td>
			<td>No</td>
			<td>ns</td>
			<td>0.1101</td>
		</tr>
		<tr>
			<td>M&#934; vs. M&#934; + IFN-&#947;</td>
			<td>16521</td>
			<td>10896 to 22145</td>
			<td>Yes</td>
			<td>****</td>
			<td>&#60;0.0001</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 minutes</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MSC/M&#934; vs. M&#934;</td>
			<td>5657</td>
			<td>32.12 to 11282</td>
			<td>Yes</td>
			<td>*</td>
			<td>0.0481</td>
		</tr>
		<tr>
			<td>MSC/M&#934; + IFN-&#947; vs. M&#934; + IFN-&#947;</td>
			<td>4932</td>
			<td>-692.9 to 10557</td>
			<td>No</td>
			<td>ns</td>
			<td>0.1079</td>
		</tr>
		<tr>
			<td>M&#934; vs. M&#934; + IFN-&#947;</td>
			<td>19083</td>
			<td>13458 to 24708</td>
			<td>Yes</td>
			<td>****</td>
			<td>&#60;0.0001</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60 minutes</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MSC/M&#934; vs. M&#934;</td>
			<td>12376</td>
			<td>6752 to 18001</td>
			<td>Yes</td>
			<td>****</td>
			<td>&#60;0.0001</td>
		</tr>
		<tr>
			<td>MSC/M&#934; + IFN-&#947; vs. M&#934; + IFN-&#947;</td>
			<td>9361</td>
			<td>3736 to 14986</td>
			<td>Yes</td>
			<td>***</td>
			<td>0.0002</td>
		</tr>
		<tr>
			<td>M&#934; vs. M&#934; + IFN-&#947;</td>
			<td>24748</td>
			<td>19123 to 30373</td>
			<td>Yes</td>
			<td>****</td>
			<td>&#60;0.0001</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70 minutes</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MSC/M&#934; vs. M&#934;</td>
			<td>13770</td>
			<td>8145 to 19395</td>
			<td>Yes</td>
			<td>****</td>
			<td>&#60;0.0001</td>
		</tr>
		<tr>
			<td>MSC/M&#934; + IFN-&#947; vs. M&#934; + IFN-&#947;</td>
			<td>11987</td>
			<td>6362 to 17612</td>
			<td>Yes</td>
			<td>****</td>
			<td>&#60;0.0001</td>
		</tr>
		<tr>
			<td>M&#934; vs. M&#934; + IFN-&#947;</td>
			<td>27264</td>
			<td>21639 to 32888</td>
			<td>Yes</td>
			<td>****</td>
			<td>&#60;0.0001</td>
		</tr>
	</tbody>
</table>
Table 3: Detailed statistical analysis of the data presented in Figure 3A. Results of Tukey&#39;s multiple comparison tests after a significant two-way ANOVA of data presented in Figure 3A.
Supplementary File 1: A representative spreadsheet file of raw kinetic data from an optimal experiment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62729/Supplementary File 1.xlsx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging at JoVE.com

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

Presented here is a protocol to quantify and produce dynamic images of mesenchymal stem cell (MSC) mediated regulation of macrophage (M&#934;) phagocytosis of non-opsonized yeast (zymosan) particles that are conjugated to a pH-sensitive fluorescent molecule.

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics ...

mesenchymal-stem-cell-regulation-macrophage-phagocytosis-quantitation

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

3.1K Views.  Molloy College. Deze co-kweekmethode zal helpen bij het beantwoorden van belangrijke vragen rond de celcontactmechanismen achter mesenchymale stamcelregulatie van macrofaagfagocytose, en daarom de rol van MSC bij het reguleren van aangeboren immuniteit belichten. Deze techniek kan worden toegepast in high-throughput analyses. De biodeeltjes geconjugeerd aan pH-gevoelige kleurstoffen fluoresceren alleen in zure fagolysoomomgevingen.Daarom zijn wassen en blussen noodzakelijk. Deze methode kan ook worden...

Video: Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Cancer Research

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a variety of particles. Normally, BMDMs are restricted from the central nervous system (CNS), but following an injury, they can readily infiltrate. Once within the injured CNS tissue, BMDMs are the primary cell type responsible for the clearance of injury-derived cellular debris, including large quantities of lipid rich myelin debris. The neuropathological ramifications of BMDM infiltration and myelin debris phagocytosis within the CNS are complex and not well understood. The protocols described here, allow for the direct in vitro study of BMDMs in the context of CNS injury. We cover murine BMDM isolation and culture, myelin debris preparation, and assays to assess BMDM myelin debris phagocytosis. These techniques produce robust quantifiable results without the need for significant specialized equipment or materials, yet can be easily customized to meet the needs of researchers.

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

We present methods to assess the phagocytic capacity of primary murine bone marrow-derived macrophages using fluorescently labeled myelin debris and intracellular lipid droplet staining.

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a ...

Neuroscience

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.

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 ...

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key players in health and disease, acting as phagocytes during immune defense and mediating trophic, maintenance, and repair functions. Although it has been possible to study some of the molecular processes involved in human macrophage function, it has proved difficult to apply genetic engineering techniques to primary human macrophages. This has significantly hampered our ability to interrogate the complex genetic pathways involved in macrophage biology and to generate models for specific disease states. An off-the-shelf source of human macrophages that is amenable to the vast arsenal of genetic manipulation techniques would, therefore, provide a valuable tool in this field. We present an optimized protocol that allows for the generation of macrophages from human induced pluripotent stem cells (iPSCs) in vitro. These iPSC-derived macrophages (iPSC-DMs) express human macrophage cell surface markers, including CD45, 25F9, CD163, and CD169, and our live-cell imaging functional assay demonstrates that they exhibit robust phagocytic activity. Cultured iPSC-DMs can be activated to different&#160;macrophage states that display altered gene expression and phagocytic activity by the addition of LPS and IFNg, IL4, or IL10. Thus, this system provides a platform to generate human macrophages carrying genetic alterations that model specific human disease and a source of cells for drug screening or cell therapy to treat these diseases.

This protocol describes the robust generation of macrophages from human induced pluripotent stem cells, and methods for their subsequent characterization. Cell surface marker expression, gene expression, and functional assays are used to assess the phenotype and function of these iPSC-derived macrophages.

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key ...

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

Medicine

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.

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

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