Name: mesenchymal stem cell regulation of macrophage phagocytosis; quantitation and imaging
Uploaded: 2021-07-16T14:00:00
Description: Watch this Scientific Journal Video about Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging at JoVE.com

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>F...

<ol>
	<li>Phinney, D. G., Prockop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+Mesenchymal+stem/multipotent+stromal+cells:+The+state+of+transdifferentiation+and+modes+of+tissue+repair--current+views.">Concise review: Mesenchymal stem/multipotent stromal cells: The state of transdifferentiation and modes of tissue repair--current views.</a> Stem Cells. 25 (11), 2896-2902 (2007).</li><li>Bernardo, M. E., Fibbe, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells:+sensors+and+switchers+of+inflammation.">Mesenchymal stromal cells: sensors and switchers of inflammation.</a> Cell Stem Cell. 13 (4), 392-402 (2013).</li><li>Tobin, L. M., Healy, M. E., English, K., Mahon, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mesenchymal+stem+cells+suppress+donor+CD4(+)+T+cell+proliferation+and+reduce+pathology+in+a+humanized+mouse+model+of+acute+graft-versus-host+disease.">Human mesenchymal stem cells suppress donor CD4(+) T cell proliferation and reduce pathology in a humanized mouse model of acute graft-versus-host disease.</a> Clinical and Experimental Immunology. 172 (2), 333-348 (2013).</li><li>Giuliani, M., 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=Long-lasting+inhibitory+effects+of+fetal+liver+mesenchymal+stem+cells+on+T-lymphocyte+proliferation.">Long-lasting inhibitory effects of fetal liver mesenchymal stem cells on T-lymphocyte proliferation.</a> PLoS One. 6 (5), 19988 (2011).</li><li>Plumas, J., Chaperot, L., Richard, M. J., Molens, J. P., Bensa, J. C., Favrot, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+induce+apoptosis+of+activated+T+cells.">Mesenchymal stem cells induce apoptosis of activated T cells.</a> Leukemia. 19 (9), 1597-1604 (2005).</li><li>Luz-Crawford, P., 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=Mesenchymal+stem+cells+generate+a+CD4+CD25+Foxp3++regulatory+T+cell+population+during+the+differentiation+process+of+Th1+and+Th17+cells.">Mesenchymal stem cells generate a CD4+CD25+Foxp3+ regulatory T cell population during the differentiation process of Th1 and Th17 cells.</a> Stem Cell Research &amp; Therapy. 4 (3), 65 (2013).</li><li>Waterman, R. S., Tomchuck, S. L., Henkle, S. L., Betancourt, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+mesenchymal+stem+cell+(MSC)+paradigm:+Polarization+into+a+pro-inflammatory+MSC1+or+an+Immunosuppressive+MSC2+phenotype.">A new mesenchymal stem cell (MSC) paradigm: Polarization into a pro-inflammatory MSC1 or an Immunosuppressive MSC2 phenotype.</a> PLoS One. 5 (4), 10088 (2010).</li><li>Nemeth, K., 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=marrow+stromal+cells+attenuate+sepsis+via+prostaglandin+E(2)-dependent+reprogramming+of+host+macrophages+to+increase+their+Interleukin-10+production.">marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host macrophages to increase their Interleukin-10 production.</a> Nature Medicine. 15 (1), 42-49 (2009).</li><li>Maggini, 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=Mouse+Bone+Marrow-Derived+Mesenchymal+Stromal+Cells+Turn+Activated+Macrophages+into+a+Regulatory-Like+Profile.">Mouse Bone Marrow-Derived Mesenchymal Stromal Cells Turn Activated Macrophages into a Regulatory-Like Profile.</a> PLoS One. 5 (2), 9252 (2010).</li><li>Takizawa, N., 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=marrow-derived+mesenchymal+stem+cells+propagate+immunosuppressive/anti-inflammatory+macrophages+in+cell-to-cell+contact-independent+and-dependent+manners+under+hypoxic+culture.">marrow-derived mesenchymal stem cells propagate immunosuppressive/anti-inflammatory macrophages in cell-to-cell contact-independent and-dependent manners under hypoxic culture.</a> Experimental Cell Research. 358 (2), 411-420 (2017).</li><li>Kim, J., Hematti, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell-educated+macrophages:+a+novel+type+of+alternatively+activated+macrophages.">Mesenchymal stem cell-educated macrophages: a novel type of alternatively activated macrophages.</a> Experimental Hematology. 37 (12), 1445-1453 (2009).</li><li>Evans, J. F., Salvador, V., George, S., Trevino-Gutierrez, C., Nunez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+aorta-derived+mesenchymal+progenitor+cells+contribute+to+and+enhance+the+immune+response+of+macrophage+cells+under+inflammatory+conditions.">Mouse aorta-derived mesenchymal progenitor cells contribute to and enhance the immune response of macrophage cells under inflammatory conditions.</a> Stem Cell Research &amp; Therapy. 6 (1), 56 (2015).</li><li>Cho, D. I., 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=Mesenchymal+stem+cells+reciprocally+regulate+the+M1/M2+balance+in+mouse+bone+marrow-derived+macrophages.">Mesenchymal stem cells reciprocally regulate the M1/M2 balance in mouse bone marrow-derived macrophages.</a> Experimental Molecular Medicine. 46, 70 (2014).</li><li>Fernandez, N., 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=Mouse+mesenchymal+progenitor+cells+expressing+adipogenic+and+osteogenic+transcription+factors+suppress+the+macrophage+inflammatory+response.">Mouse mesenchymal progenitor cells expressing adipogenic and osteogenic transcription factors suppress the macrophage inflammatory response.</a> Stem Cells International. 2017, 5846257 (2017).</li><li>Jackson, M. 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>

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 phagocy...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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)&#160;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&#160;cm2, 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&#934; 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-&#947;</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 &#181;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 ...

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 ...

This co-culture method will help answer key questions surrounding the cell contact mechanisms behind mesenchymal stem cell regulation of macrophage phagocytosis, and therefore illuminate the role of MSC in regulating innate immunity. This technique can be applied in high-throughput analyses. The bioparticles conjugated to pH-sensitive dyes fluoresce only in acidic phagolysosome environments.Hence, washing and quenching are necessary. This method can also be applied to a variety of co-culture models that include neutrophils and other phagocytes. Demonstrating the procedure will be Ethan Chetkof, an undergraduate currently in my lab.To begin with, observe the confluency of cultured mesenchymal stem cells. When 70-80%confluency is achieved, aspirate the growth medium from the cultured cells in a 100 millimeter dish and add five milliliters of PBS. After aspirating the PBS, add two milliliters of 0.05%trypsin EDTA solution and incubate the cells for three minutes.After three minutes, check the cell detachment using an inverted microscope. And if the cells are not detached, incubate for one to two minutes again. Once all the cells are detached, add five milliliters of fresh growth medium to the plate.After rinsing the plate using the trypsin medium mixture, use a sterile serological pipette to collect cells into a clean, sterile 50 milliliter conical tube. Next, pellet down the cells by centrifugation. After aspirating the supernatant, resuspend the cell pellet in 10 milliliters of fresh growth medium by gently pipetting up and down.For cell counting, add 10 microliters of cell suspension into a 1.5 milliliter microcentrifuge tube containing 30 microliters of 0.4%Trypan Blue solution, then add 10 microliters of the mixture under the coverslip of a hemocytometer counting chamber. Under the inverted or Brightfield microscope, count the unstained viable cells using a 10X objective lens and four one-square millimeter chambers and calculate the cell number as described in the text manuscript. Use the equation C1V1 equals C2V2 to determine the desired volume of the fresh medium and cell suspension required to prepare the cell suspension at a final concentration of one times 10 to the fifth cells per milliliter.Use a multi-channel micropipetter to add 100 microliters of the cell suspension in the wells of a 96-well plate according to the plate design. Use a micropipette to add 530 microliters of the cell suspension to each one of the chamber slide according to the plate design and incubate overnight. Prepare 10 milliliters of activation medium containing interferon gamma at a concentration of 250 nanograms per milliliter for activating the macrophage cells in one 100-millimeter dish.Aspirate the growth medium from the macrophage cell cultures and rinse the cells with five milliliters of the activation medium devoid of interferon gamma. After aspirating the rinsing medium in the experimental dish, add 10 milliliters of activation medium supplemented with interferon gamma. And in the control dish, add 10 milliliters of activation medium without interferon gamma, then incubate the cells for 16 to 24 hours.At the end of the incubation, for preparing the co-cultures, remove the activation medium from the macrophage cells and add five milliliters of fresh growth medium. Use a cell lifter to gently scrape the cells and collect the cells into a 50 milliliter conical tube. Next, count the cells using Trypan Blue exclusion and hemocytometry, followed by preparation of control and treated macrophage cell suspensions at the concentration of two times 10 to the fifth cells per milliliter as demonstrated previously for the mesenchymal stem cells.Gently aspirate the medium from the experimental wells containing mesenchymal stem cells. Use a multi-channel micropipette to add 100 microliters of the treated and the control macrophage cell suspensions in the appropriate experimental wells according to the plate design and incubate overnight as demonstrated. Gently aspirate the medium from the four-walled chamber slide containing mesenchymal stem cells.Use a micropipette to add 530 microliters of the macrophage cell suspension to the appropriate wells according to the design and incubate overnight. Add one milliliter of live cell imaging medium to the vial containing one milligram of Zymosan particles and collect the medium particle mixture into a glass culture tube. After rinsing the vial with an additional one milliliter of imaging solution, transfer the rinsed imaging medium in the glass tube, then add three milliliters of additional imaging solution to achieve 0.2 milligrams per milliliter Zymosan particle suspension.Vortex the Zymosan suspension using quick pulses for 30 to 60 seconds, then use a probe sonicator to sonicate the suspension with 60 quick pulses. After aspirating the medium, rinse the experimental wells and reagent blank wells with 100 microliters of live cell imaging solution. Next, aspirate the live cell imaging solution from the wells and add 100 microliters of the Zymosan suspension.Open the plate tray of the fluorescent reader using the touch pad interface. Set the plate without the lid in the tray with the A1 well in the upper left corner. Close the tray using the touch pad and click on the green read button in the top menu.To conduct the phagocytosis assay, after rinsing the first experimental well with 750 microliters of the imaging medium, add 400 microliters of the Zymosan suspension leaving the remaining wells in the growth medium. Next, place the slide on the incubated stage of the imaging system. Use the imaging software.Select the Brightfield option under the locate tab, and then click on the eyepiece icon. With the 10X objective in place, use the eyepiece and focusing knob to focus on the cells. Select the acquisition tab.Open the experiment dropdown menu and select a set of wavelengths that includes EGFP filter set to accommodate 509 nanometers excitation and 533 nanometers emission wavelengths of the pH-sensitive dye. When about two minutes are left on the timer, click on the 20X icon in the objective menu to change the objective to 20X, then click on the channels menu, select EGFP filters, and in the exposure menu, set the exposure time to 400 milliseconds to detect the pH-sensitive green fluorophore, and then click on live. After adjusting the focus, click on stop to turn off the light and check the box next to the top time-lapse experimental setting.In the focusing strategy menu, select software auto-focus and from the dropdown menu, select smart and core settings to reduce the time of exposure to light while the auto-focus is running. In the time-lapse menu, set the desired number of acquisitions to 30 to 60, the interval time to one minute, and then click on the start experiment button to begin the acquisition. The effect of interferon gamma on the co-culture was studied.The representative results demonstrate that the co-culture of macrophages with mesenchymal stem cells enhances the phagocytic activity of the macrophage. Interferon gamma treatment reduces the activity of the macrophage. However, in the presence of mesenchymal stem cells, the macrophage phagocytic activity was partially rescued.The optimal cell densities are critical in these studies. And when the macrophage cells were plated at too low of a density, changes in fluorescence intensity were not detected. When the macrophage cells were plated at a high density, the fluorescence intensity was elevated rapidly in all groups and differences were not discerned.One of the most important things during this procedure is to ensure that the cell densities are optimized and kept consistent between experiments.

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

Research

JoVE Journal

Immunology and Infection

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

The immunomodulatory properties of human mesenchymal stem cells (MSC) appear increasingly relevant for clinical application. Using a co-culture system of MSCs and peripheral blood leukocytes pre-stained with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE), we describe the in vitro assessment of MSC immunomodulation on effector leukocyte proliferation and specific subpopulations.

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein. They actively uptake saturated and unsaturated fatty acids, which might play a critical role in regulating their immune functions. Numerous studies have shown that various fatty acids, saturated or unsaturated, may possess different impacts on cell growth and function. However, the approaches used for fatty acid preparation vary, which may lead to non-physiological results. Serum albumin, a natural carrier for fatty acids in mammalian peripheral blood, is recommended for forming a conjugate complex with the sodium salt of fatty acids to study fatty acid function in mammalian cells, thus minimizing the toxicity of fatty acid soap. Thus, a simple, relatively quick heating and sonicating method is developed and presented here for BSA-fatty acid conjugate formation. We describe a protocol using saturated fatty acids, especially stearic acids to induce severe cell death in mouse bone-marrow derived macrophages. We further demonstrate that saturated fatty acid-induced cell death is positively associated with accumulated cellular ceramide levels. This method can be extended for studies of the impact of fatty acid on other mammalian cells.

We illustrate a straight-forward method to derive murine primary macrophages from bone marrow cells and a simple method to prepare BSA-fatty acid conjugates. Then we demonstrate that saturated fatty acids can induce macrophage cell death, and such cell death is positively associated with cellular accumulation of ceramide levels.

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein. They actively uptake saturated and unsaturated ...

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 ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.