Name: assessment of antibody-based drugs effects on murine bone marrow and peritoneal macrophage activation
Uploaded: 2017-12-26T15:00:00
Description: Watch this Scientific Journal Video about Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation at JoVE.com

L.K. is the recipient of the University of British Columbia 4-year fellowship (4YF) graduate studentship award. L.M.S. is the recipient of a Canadian Association of Gastroenterology/ Crohn's and Colitis Canada/ CIHR New Investigator Salary Award and is a Biomedical Scholar of the Michael Smith Foundation for Health Research. This work was supported by a Project Grant from Canadian Blood Services, in collaboration with the Canadian Institutes of Health Research (grant # CIHR2016-LS).

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of British Columbia.
1. Bone Marrow Macrophage Derivation and Activation with Antibodies
<ol>
	<li>Perform euthanasia using CO2 asphyxiation.
	<ol>
		<li>Place mouse in induction chamber. Euthanize mouse with 5% isoflurane anesthesia, for 60 - 90 s, until immobile and breathing is deep and slow.</li>
		<li>Turn off isoflurane anesthesia and administer 6 - 8 L/...

<ol>
	<li>Murray, P. J., Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+and+pathogenic+functions+of+macrophage+subsets.">Protective and pathogenic functions of macrophage subsets.</a> Nat Rev Immunol. 11 (11), 723-737 (2011).</li><li>Martinez, F. O., Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+M1+and+M2+paradigm+of+macrophage+activation:+time+for+reassessment.">The M1 and M2 paradigm of macrophage activation: time for reassessment.</a> F1000Prime Rep. 6, 13 (2014).</li><li>Mosser, D. M., Edwards, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+full+spectrum+of+macrophage+activation.">Exploring the full spectrum of macrophage activation.</a> Nat Rev Immunol. 8 (12), 958-969 (2008).</li><li>Edwards, J. P., Zhang, X., Frauwirth, K. A., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+functional+characterization+of+three+activated+macrophage+populations.">Biochemical and functional characterization of three activated macrophage populations.</a> J Leukoc Biol. 80 (6), 1298-1307 (2006).</li><li>Mosser, D. M., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+murine+macrophages.">Activation of murine macrophages.</a> Curr Protoc Immunol. , (2008).</li><li>Gallo, P., Gon&#231;alves, R., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+IgG+density+and+macrophage+Fc+(gamma)+receptor+cross-linking+on+phagocytosis+and+IL-10+production.">The influence of IgG density and macrophage Fc (gamma) receptor cross-linking on phagocytosis and IL-10 production.</a> Immunol Lett. 133 (2), 70-77 (2010).</li><li>Vos, A. C., 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=Anti-tumor+necrosis+factor-&#945;+antibodies+induce+regulatory+macrophages+in+an+Fc+region-dependent+manner.">Anti-tumor necrosis factor-&#945; antibodies induce regulatory macrophages in an Fc region-dependent manner.</a> Gastroenterology. 140 (1), 221-230 (2011).</li><li>Kozicky, L. 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=Intravenous+immunoglobulin+skews+macrophages+to+an+anti-inflammatory,+IL-10-producing+activation+state.">Intravenous immunoglobulin skews macrophages to an anti-inflammatory, IL-10-producing activation state.</a> J Leukoc Biol. 98 (6), 983-994 (2015).</li><li>Sutterwala, F. S., Noel, G. J., Salgame, P., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+proinflammatory+responses+by+ligating+the+macrophage+Fcgamma+receptor+type+I.">Reversal of proinflammatory responses by ligating the macrophage Fcgamma receptor type I.</a> J Exp Med. 188 (1), 217-222 (1998).</li><li>Nimmerjahn, F., Ravetch, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antiinflammatory+activity+of+IgG:+the+intravenous+IgG+paradox.">The antiinflammatory activity of IgG: the intravenous IgG paradox.</a> J Exp Med. 204 (1), 11-15 (2007).</li><li>Gelfand, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+immune+globulin+in+autoimmune+and+inflammatory+diseases.">Intravenous immune globulin in autoimmune and inflammatory diseases.</a> N Engl J Med. 368 (8), 777 (2013).</li><li>Andersson, J., Skans&#233;n-Saphir, U., Sparrelid, E., Andersson, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+immune+globulin+affects+cytokine+production+in+T+lymphocytes+and+monocytes/macrophages.">Intravenous immune globulin affects cytokine production in T lymphocytes and monocytes/macrophages.</a> Clin Exp Immunol. 104, 10-20 (1996).</li><li>Saito, S., Matsuura, M., Hirai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+lipopolysaccharide-induced+interleukin-12+production+by+activation+of+repressor+element+GA-12+through+hyperactivation+of+the+ERK+pathway.">Regulation of lipopolysaccharide-induced interleukin-12 production by activation of repressor element GA-12 through hyperactivation of the ERK pathway.</a> Clin Vaccine Immunol. 13 (8), 876-883 (2006).</li><li>Cao, S., Zhang, X., Edwards, J. P., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF-kappaB1+(p50)+homodimers+differentially+regulate+pro-+and+anti-inflammatory+cytokines+in+macrophages.">NF-kappaB1 (p50) homodimers differentially regulate pro- and anti-inflammatory cytokines in macrophages.</a> J Biol Chem. 281 (36), 26041-26050 (2006).</li><li>Neurath, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+targets+for+mucosal+healing+and+therapy+in+inflammatory+bowel+diseases.">New targets for mucosal healing and therapy in inflammatory bowel diseases.</a> Mucosal Immunol. 7 (1), 6-19 (2014).</li><li>Bryant, A., Moore, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rituximab+and+its+potential+for+the+treatment+of+rheumatoid+arthritis.">Rituximab and its potential for the treatment of rheumatoid arthritis.</a> Ther Clin Risk Manag. 2 (2), 207-212 (2006).</li><li>Weiner, L. M., Surana, R., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies:+versatile+platforms+for+cancer+immunotherapy.">Monoclonal antibodies: versatile platforms for cancer immunotherapy.</a> Nat Rev Immunol. 10 (5), 317-327 (2010).</li><li>Louis, K. S., Siegel, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+viability+analysis+using+trypan+blue:+manual+and+automated+methods.">Cell viability analysis using trypan blue: manual and automated methods.</a> Methods Mol Biol. 740, 7-12 (2011).</li><li>Liu, Z. Q., Mahmood, T., Yang, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blot:+technique,+theory+and+trouble+shooting.">Western blot: technique, theory and trouble shooting.</a> N Am J Med Sci. 6 (3), 160 (2014).</li><li>Nolan, T., Hands, R. E., Bustin, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+mRNA+using+real-time+RT-PCR.">Quantification of mRNA using real-time RT-PCR.</a> Nat Protoc. 1 (3), 1559-1582 (2006).</li><li>Lucas, M., Zhang, X., Prasanna, V., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ERK+activation+following+macrophage+FcgammaR+ligation+leads+to+chromatin+modifications+at+the+IL-10+locus.">ERK activation following macrophage FcgammaR ligation leads to chromatin modifications at the IL-10 locus.</a> J Immunol. 175 (1), 469-477 (2005).</li><li>Murray, P. 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=Macrophage+activation+and+polarization:+nomenclature+and+experimental+guidelines.">Macrophage activation and polarization: nomenclature and experimental guidelines.</a> Immunity. 41 (1), 14-20 (2014).</li><li>Anderson, C. F., Gerber, J. S., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+macrophage+function+with+IgG+immune+complexes.">Modulating macrophage function with IgG immune complexes.</a> J Endotoxin Res. 8 (6), 477-481 (2002).</li><li>Sutterwala, F. S., Noel, G. J., Clynes, R., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+suppression+of+interleukin-12+induction+after+macrophage+receptor+ligation.">Selective suppression of interleukin-12 induction after macrophage receptor ligation.</a> J Exp Med. 185 (11), 1977-1985 (1997).</li><li>Riquelme, 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=IFN-&#947;-induced+iNOS+expression+in+mouse+regulatory+macrophages+prolongs+allograft+survival+in+fully+immunocompetent+recipients.">IFN-&#947;-induced iNOS expression in mouse regulatory macrophages prolongs allograft survival in fully immunocompetent recipients.</a> Mol Ther. 21 (2), 409-422 (2013).</li><li>Vos, A. C., 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=Regulatory+macrophages+induced+by+infliximab+are+involved+in+healing+in+vivo+and+in+vitro.">Regulatory macrophages induced by infliximab are involved in healing in vivo and in vitro.</a> Inflamm Bowel Dis. 18 (3), 401-408 (2012).</li><li>Zhang, X., Goncalves, R., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+and+characterization+of+murine+macrophages.">The isolation and characterization of murine macrophages.</a> Curr Protoc Immunol. , (2008).</li><li>Gerber, J. S., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversing+lipopolysaccharide+toxicity+by+ligating+the+macrophage+Fc+gamma+receptors.">Reversing lipopolysaccharide toxicity by ligating the macrophage Fc gamma receptors.</a> J Immunol. 166 (11), 6861-6868 (2001).</li><li>Durandy, A., 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=Intravenous+immunoglobulins--understanding+properties+and+mechanisms.">Intravenous immunoglobulins--understanding properties and mechanisms.</a> Clin Exp Immunol. 158, 2-13 (2009).</li><li>Jolles, S., Sewell, W. A., Misbah, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+uses+of+intravenous+immunoglobulin.">Clinical uses of intravenous immunoglobulin.</a> Clin Exp Immunol. 142 (1), 1-11 (2005).</li><li>Murakami, 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=Intravenous+immunoglobulin+preparation+prevents+the+production+of+pro-inflammatory+cytokines+by+modulating+NF&#954;B+and+MAPKs+pathways+in+the+human+monocytic+THP-1+cells+stimulated+with+procalcitonin.">Intravenous immunoglobulin preparation prevents the production of pro-inflammatory cytokines by modulating NF&#954;B and MAPKs pathways in the human monocytic THP-1 cells stimulated with procalcitonin.</a> Inflamm Res. 63 (9), 711-718 (2014).</li></ol>

Macrophage activation states play an important role in both tissue homeostasis and disease22. Macrophages can have distinct as well as overlapping activation states, depending on cues in their microenvironment3. They have distinct roles in all stages of the inflammatory response: defense against pathogens, wound healing and tissue restitution, and also have a distinct anti-inflammatory activation state that is important for turning off the inflammatory response<sup class="x...

Macrophages are innate immune cells, which play multiple roles in the immune response to infection or injury. Macrophages are responsible for initiating an immune response to infection or tissue damage, stopping the inflammatory response, and promoting the healing response1. Examples of the three best studied macrophage activation states are: 1) macrophages treated with interferon gamma (IFN&#947;) and bacterial lipopolysaccharide (LPS), designated M(IFN&#947; + LPS), which contribute to the inflammatory response; 2) macrophages stimulated with interleukin 4 (IL-4), M(IL-4), which are associated with the healing response; 3) macrophages stimulated with immune complexes (IC) and LPS, M(IC + LPS), which have the ability to turn off the inflammatory response2,3. M(IC + LPS) are distinct from M(IL-4) wound healing macrophages, and do not express the enzyme arginase (Arg-1) or FIZZ14. The best marker for these anti-inflammatory macrophages is their cytokine production5. Macrophages have multiple roles in maintaining health, but also contribute to inflammatory diseases and cancer3. Because of this, macrophages are a key therapeutic target for the treatment of a wide variety of diseases. It is important to investigate the effects of antibodies on their activation state to develop macrophage-based treatments for disease.
The focus of this paper is on the use of murine bone marrow derived macrophages (BMDMs) and peritoneal macrophages to test the effect of antibody drugs on inflammatory responses in vitro and in vivo. Recently, there have been multiple studies showing the effects of antibodies on macrophage activation6,7,8. Macrophages co-activated with immune complexes, which are antibodies complexed with an antigen, and LPS, a normally inflammatory stimulus, produce very high levels of the anti-inflammatory cytokine, IL-10, and very low levels of the pro-inflammatory cytokine, interleukin 12 (IL-12)9. In addition, infliximab, a monoclonal antibody against TNF&#945;, has been found to work, in part, by inducing anti-inflammatory macrophages through its fragment crystallizable (Fc) region7. We have reported that IVIg + LPS induce anti-inflammatory macrophage activation that is similar to M(IC + LPS), wherein co-stimulated macrophages produce large amounts of IL-10 and low amounts of the pro-inflammatory cytokine subunit Interleukin 12 or 23 p40 (IL-12/23p40), interleukin- 6 (IL-6), and TNF8. IVIg is a drug comprised of polyclonal antibodies, primarily IgG, which has been pooled from the blood of more than 1,000 donors10. It is used to treat a wide variety of immunological diseases, such as idiopathic thrombocytopenic purpura and chronic demyelinating polyneuropathy, but its mechanism of action is not completely understood11. The effects of antibody based drugs on macrophage activation can be assessed using methods described herein.
The effects of specific biologics on macrophage activation can be tested in BMDMs and peritoneal macrophages. Using these macrophage sources permits assessment of primary cells. Preliminary testing of antibodies on cultured primary cells requires less time and monetary investment than other time consuming and expensive disease models. By injecting a drug into a healthy mouse in vivo, and isolating the cells and analyzing them ex vivo, one can determine if studies are warranted to assess whether treatment with biologics affects macrophage activation in disease models.
With few studies testing the effect of biologic therapies on macrophage activation in vitro and in vivo directly, our techniques provide an advantage over alternative techniques. Current techniques involve testing biologic drug effects on mixed cell populations in vitro, such as the effect of infliximab in a mixed lymphocyte reaction (MLR) or IVIg on human macrophages in peripheral blood mononuclear cells, where the effect cannot be attributed to a specific cell type7,12. Using BMDMs and peritoneal macrophages is advantageous over using cell lines, such as RAW264.7 cells, which do not produce the pro-inflammatory cytokine, IL-12, in response to LPS8,13. Testing the effect of an antibody-based drug on macrophage responses ex vivo has advantages because cytokine responses can be attributed directly to macrophages, rather than inferring macrophage responses by measuring serum cytokine levels14. BMDMs and peritoneal macrophages can be derived and isolated from genetically modified mice to determine the specific role of a protein in anti-inflammatory macrophage activation. For example, we have used Il10 deficient(-/-) BMDMs to demonstrate that IVIg-induced reduction of pro-inflammatory cytokine production is partially dependent on IL-108. A drug&#39;s mechanism of action can be investigated using western blotting, where the role of specific proteins and signaling events can be determined. Quantitative polymerase chain reaction (qPCR) can be performed on BMDMs or peritoneal macrophages to show patterns of gene expression that result from antibody activation. Disease models in mice can provide information on the potential efficacy of antibody-based biological therapies in models for diseases like inflammatory bowel disease, rheumatoid arthritis, and cancer15,16,17. However, the techniques described here will provide information on the mechanism of action of these biologics by determining whether they induce anti-inflammatory macrophage activity.

<table border="0" cellpadding="0" cellspacing="0" style="width:1312px;" width="1311">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Iscove&#8217;s modified Dulbecco&#8217;s medium (IMDM)</td>
			<td style="width:209px;">Life technologies</td>
			<td style="width:408px;">12440053</td>
			<td style="width:241px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:209px;">Life technologies</td>
			<td style="width:408px;">12483-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Recombinant murine macrophage colony stimulating factor (MCSF)</td>
			<td style="width:209px;">Stemcell technologies</td>
			<td>78059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Penicillin-streptomycin</td>
			<td style="width:209px;">Life technologies</td>
			<td style="width:408px;">15140148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Monothioglycerol (MTG)</td>
			<td style="width:209px;">Sigma</td>
			<td>88640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">1X red blood cell lysis buffer</td>
			<td style="width:209px;">eBioscience</td>
			<td style="width:408px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Cell dissociation buffer</td>
			<td style="width:209px;">Life technologies</td>
			<td style="width:408px;">13150016</td>
			<td>Enzyme-Free, Hanks&#39;s-based, EDTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Lipopolysaccharide (LPS)</td>
			<td style="width:209px;">Sigma aldrich</td>
			<td style="width:408px;">L 4516</td>
			<td>From E. coli 0127:B8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">IVIg (Gammunex)</td>
			<td style="width:209px;">Grifols</td>
			<td>Received from BC Children&#39;s Hospital, Transfusion Medicine</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">IVIg (Gammagard liquid)</td>
			<td style="width:209px;">Baxter Healthcare Corporation</td>
			<td>Received from BC Children&#39;s Hospital, Transfusion Medicine</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">IVIg (Octagam)</td>
			<td style="width:209px;">Octapharma</td>
			<td>Received from BC Children&#39;s Hospital, Transfusion Medicine</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Phosphate buffered saline (PBS) (sterile), pH 7.4</td>
			<td style="width:209px;">Life technologies</td>
			<td style="width:408px;">10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">mouse IL-10 ELISA</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">555252</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">mouse IL-12/23p40 ELISA</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">555165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">anti-Erk1/2 antibody</td>
			<td style="width:209px;">Cell signalling technology</td>
			<td style="width:408px;">9101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">anti-pp38 antibody</td>
			<td style="width:209px;">Cell signalling technology</td>
			<td style="width:408px;">9211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">anti-GAPDH antibody</td>
			<td style="width:209px;">Fitzgerald industries</td>
			<td style="width:408px;">10R-G109A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">26 g needle</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">305110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">1 mL syringe</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">10 mL syringe</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">309604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">15 mL conical tube</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">352096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">50 mL conical tube</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">microcentrifuge tube (1.7 mL)</td>
			<td style="width:209px;">Diamed</td>
			<td style="width:408px;">SPE155-N</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:453px;">75 cm2 tissue culture treated flask</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">353136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Cell scraper</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">353085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Forcepts</td>
			<td style="width:209px;">VWR</td>
			<td>82027-386</td>
			<td>fine tip, dissecting&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:453px;">Scissors</td>
			<td style="width:209px;">VWR</td>
			<td>82027-582</td>
			<td>Delicate, 4 1/2&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Brightfield microscope</td>
			<td style="width:209px;">Motic</td>
			<td style="width:408px;">AE31</td>
			<td>Inverted phase contrast</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Scale&#160;</td>
			<td style="width:209px;">Mettler&#160;</td>
			<td style="width:408px;">PE 3000</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of antibody-based drugs effects on murine bone marrow and peritoneal macrophage activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Murine bone marrow derived macrophages can be cultured from hematopoietic cell precursors in bone marrow aspirates. Bone marrow aspirates pooled from femurs and tibias of one C57BL/6 mouse typically yield 107 bone marrow derived macrophages, making them a convenient source of macrophages for experiments. BMDMs can be used to test antibody based drug responses when challenged with an inflammatory stimulus in vitro. Figure 1 shows that IVIg ...

Watch this Scientific Journal Video about Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation at JoVE.com

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

The overall goal of these experiments is to determine the effects of antibody-based drugs on anti-inflammatory macrophage activation states using mouse bone marrow and peritoneal macrophages. This method can help answer key questions in the field of immunology, such as, do specific antibody drugs act by changing macrophages to an IL-10 producing anti-inflammatory activation state. The main advantage of this technique, is that it measures the response of primary mouse macrophages from the bone marrow in vitro and peritoneal cavity in vivo.Perform this procedure in a sterile hood. Secure a euthanized mouse supine on a foam board with its limbs outstretched. Then, clean the legs with 70%ethanol.Next, make a shallow cut about two millimeters deep to remove the skin and fur from the surface of each hind leg. Then, dissect the tissues to access the tibias and femurs. To remove the tibias and femurs, cut the bone and muscles just below the hip joint, and above the ankles.Then, remove as much of the muscle tissue from the bones as possible. Next, clean the bones with 70%ethanol, and allow it to evaporate for one minute. Then, place the bones in a six well plate filled with bone flesh medium.In the plate, trim one millimeter from either end of the femurs so that their inner cavities are exposed. Make certain a 26-gauge needle can access the cavities. Next, separate the tibia and femur from the knee joint.Then, insert the needle attached to a 10 milliliter syringe into a bone cavity and collect the bone marrow. Pull the bone marrow from all four bones into a 50 milliliter conical tube with five milliliters of bone flesh medium. Pipe out the medium and marrow up and down several times.Or vortex at a slow speed to gently disperse clumps in the suspension. Strings of marrow should be broken up into small flecks of marrow less than half a millimeter long. Next, top off the tube to 50 milliliters with bone flesh medium, and transfer it into a 75 square centimeter tissue culture flask.Incubate the bone marrow for an hour. During the incubation, the matured cells will adhere to the flask, and the desired hematopoietic progenitors will remain in suspension. Transfer the suspension to a 50 milliliter conical tube and spin down the cells into a pellet.Next, discard the supernatant and resuspend the cell pellet in five milliliters of MCSF culture medium. Then adjust the cell density to 500, 000 per milliliter and load 30 milliliters into a new 75 square centimeter flask. Continue culturing the cells, and on days four, seven, and 10, verify that the cells are adherent and slightly branched using an inverted phase contrast breakfield microscope.When checking the cells, discard the medium containing the non-adherent cells. Then wash the adherent cells with IMDM one time, and add 30 milliliters of fresh MCSF culture medium back into the flask. On day 10, replace the medium with eight milliliters of enzyme-free EDTA-based cell dissociation buffer to collect the cells.Incubate the cells briefly, then scrape the cells gently, and verify that they've released using a microscope. Once the cells are released, transfer the buffer with the cells to a 50 milliliter conical tube. Then rinse the flask three times with five milliliters of IMDM to collect more cells.Pull all the collections, centrifuge them and resuspend the pellet in three milliliters of MCSF. Now, count the cells and adjust their concentration to one million per milliliter. Then plate 100 microliters of cell suspension per well in a 96-well flat bottom plate.The entire plate should be fillable. Culture the cells for about an hour until they are adherent. Then stimulate them with different concentrations of LPS or intravenous immunoglobulin or both prepared in IMDM.Perform all the manipulations in duplicate or triplicate and be certain to keep untreated controls. Then incubate the cells for 24 hours and analyze the supernatants. Prepare a one milliliter syringe with the required amount of intravenous immunoglobulin and LPS, and attach a 26-gauge needle.For controls, use PBS plus LPS instead. Take hold of the mouse by its scruff while securing its hindquarters and tail using one hand. Target the lower-right quadrant of the abdomen, and insert the needle at a 30 to 40-degree angle with the bevel up, and insert it about 1.5 centimeters.Then inject the bolus. After an hour, harvest peritoneal macrophages after euthanizing the mouse. In a sterile hood, pin the euthanized mouse to a foam board with limbs spread out and sterilize the abdomen with 70%ethanol.Then, make a shallow two millimeter incision along the midline, and pull off the abdominal skin. Avoid puncturing the peritoneal wall. Next, fully load a five milliliter syringe with sterile PBS at pH 7.4, and inject the mouse using a 25 or 27-gauge needle, at the top of the peritoneal cavity, from either side, toward the midline, with the bevel of the needle up.After pulling the needle out, massage the peritoneum for 10 seconds to dislodge the cells. Now, insert the needle again, back into the cavity, and collect about 3.75 milliliters of lavage fluid. Transfer the lavage fluid to a 15 milliliter conical tube on ice.Repeat the injection, massage, and collection of lavage fluid three more times, and pull all the collections into the same tube. During the final collection it may be easier to withdraw the lavage fluid from the far side of the mouse. Next spin down the cells, and resuspend them in 500 microliters of plating medium.Then count the viable macrophages, and resuspend them at one million per milliliter. Now, plate 100 microliters of suspension into the wells of a 96-well flat bottom plate. Next, incubate the cells for an hour.The peritoneal macrophages will adhere to the plate. Then, remove the non-adherent cells, and rinse each well twice with 200 microliters of warm IMDM. After each wash, wait 10 seconds, and slowly tilt the plate to pull the rinse solution.After the washes, add back 100 microliters of plating medium, and place the plate in the incubator. For the ELISA, continue to incubate the cells without stimulation for 24 hours. Then collect and clarify the conditioned medium for cytokine analysis.BMDMs can be used to test antibody-based drug responses when challenged with an inflammatory stimulus in vitro. Intravenous immunoglobulin with LPS increases the production of the anti-inflammatory cytokine IL-10, seven-fold, compared to LPS stimulation alone. And it decreases the production of IL-12/23p40.In addition, intravenous immunoglobulin stimulation alone or with LPS increased the activation of the MAP kinase ERK1/2, with early and prolonged phosphorylation. Activation of P-38 also occurred sooner. Furthermore, peritoneal macrophages significantly increased IL-10 production with stimulation in vitro, when compared to PBS-injected mice.Cytokine production was next assessed in lavage fluid and in supernatants from ex vivo stimulated peritoneal macrophages. IL-10 was increased in lavage fluid, peritoneal cells, and peritoneal macrophages when the mice are challenged with intravenous immunoglobulin and LPS. By comparison, production of the pro inflammatory cytokine subunit IL-12/23p40 was significantly reduced in cultured peritoneal macrophages, but not in lavage fluid.After watching this video, you should have a good understanding of how to test the effects of antibody-based drugs on macrophage activation states in vitro and in vivo using mouse bone marrow and peritoneal macrophages.

assessment-antibody-based-drugs-effects-on-murine-bone-marrow

Research

JoVE Journal

Immunology and Infection

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

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 ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...