Authors were funded in part by the intramural program of NIDCR (N.M.M) and supported by a Wellcome Trust Stepping Stones Fellowship (097820/Z/11/B to J.E.K) and by a Manchester Collaborative Centre for Inflammation Research grant (to J.E.K). The authors thank Teresa Wild for critically reviewing the manuscript.

All experimental procedures described in this protocol followed required guidelines and were approved by the Institutional Animal Care and Use Committee, NIDCR/NIH.
1. Prepare in Advance
<ol>
	<li>Prepare Complete media: RPMI supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 ug/ml streptomycin and 10% FBS.</li>
	<li>Prepare DNase media: 50 ml of RPMI media supplemented with 7.5 &#181;g DNase (Make fresh, keep on ice at all times).</li>
	<li>Prepare Collagenase-DNase media: 5 ml of DNase media plus 3.2 mg/ml of Collagenase Type IV (Make fresh, keep on ice at all times).</li>
	<li>Pre-cool FACS buffer: 0.5 % FBS diluted in PBS.</li>
	<li>Pre-cool centrifuge to 4 &#176;C.</li>
	<li>Warm shaker incubator to 37 &#176;C.</li>
</ol>
2. Isolation, Dissection, and Cell Isolation from Gingival Blocks
<ol>
	<li>Euthanize mice by exposing them to CO2 in an appropriate chamber, according to ARAC guidelines.</li>
	<li>Open the thoracic and abdominal cavity to expose the heart and internal organs. To perfuse, make an incision in the inferior vena cava and perfuse 3 ml of PBS via the left ventricle using a 3 ml syringe with a 27 G needle.</li>
	<li>Immobilize the body and head of the mouse on a pad with stomach facing up.</li>
	<li>Cut each commissure of the lip towards the neck with scissors, separating the skin that covers mandible and neck region, exposing the underlying tissues and muscles. Dissect the lower lip to uncover the mandible.</li>
	<li>Cut between the lower incisors to separate both sides of the mandible and immobilize each side to access the oral cavity.</li>
	<li>Visualize and dissect the palate away from the nasal cavity.</li>
	<li>Dissect areas using a 10 blade and include maxillary and mandibular molar areas with surrounding gingiva (Figure 1). Make vertical incisions just anterior to the first molar and posterior to the third molar and a horizontal incision at the border of the gingiva (white collar of tissue surrounding teeth).</li>
	<li>Place the maxillary and mandibular blocks in a 50 ml conical tube with 5 ml Collagenase/DNase (keep on ice).</li>
	<li>Incubate in a shaker incubator at 37 &#176;C for 1 hr and during the last 5 min of incubation add 50 &#181;l of 0.5 M EDTA.</li>
	<li>Add 5 ml of cold DNase media to the 50 ml tube with tissues and gently swirl to mix.</li>
	<li>Transfer the 4 blocks of tissue on a petri dish and cover with 500 &#181;l of DNase media. Remove gingiva from each block of tissue, using a scalpel blade and pointing the blade into the gingival and interdental areas.</li>
	<li>Transfer the tissues and the media from the petri dish as well as the previous DNase media used during dissection, to a new 50 ml tube through the cell strainer (70 &#181;m size). Wash with DNase media (3-5 ml) all of the instruments used and filter.</li>
	<li>Using the plunger of a sterile 3 ml syringe, mash the tissue against the strainer, filtering with cold DNase media (30 to 35 ml).</li>
	<li>Wash the cell strainer with cold DNase media.</li>
	<li>Centrifuge at 4 &#176;C, 314 x g for 6 min.</li>
	<li>Discard the supernatant, re-suspend in 1 ml of complete media.</li>
	<li>Count cells (the expected amount should range between 8 x 105 to 1.2 x 106 cells with a viability of &#62;80%) using an automated cell counter.</li>
</ol>
3. Stimulation and FACS Staining of Gingival Cells
<ol>
	<li>Stimulate the single cell suspension with PMA (50 ng/ml) and Ionomycin (2.5 &#181;g/ml) in the presence of 1 &#181;l/ml of Brefeldin A.</li>
	<li>Incubate for 3.5 hr at 37 &#176;C and 5% CO2.</li>
	<li>Spin down and wash with PBS and centrifuge at 4 &#176;C; 314 x g for 6 min.</li>
	<li>Stain the cells using a viability stain following the manufacturer&#39;s instructions.</li>
	<li>Stain for extracellular markers (CD45, TCR&#947;&#948;, TCR&#946;, CD4, CD8, TCR&#947;&#948; or antibodies of choice) following the manufacturer&#39;s instructions.</li>
	<li>Wash the cells with cold FACS buffer and centrifuge at 4 &#176;C, 314 x g for 5 min.</li>
	<li>Gently vortex to disrupt the cell pellet.</li>
	<li>Fix the cells with 2% paraformaldehyde for 20 min at room temperature.</li>
	<li>Wash the cells with cold FACS buffer, centrifuge at 4 &#176;C, 314 x g for 5 min and stain for intracellular cytokines.
	<ol>
		<li>Fill the FACS tubes with a solution of cold 0.5% saponin diluted in FACS buffer and centrifuge at 4 &#176;C, 314 x g for 5 min.</li>
		<li>Add 300 &#181;l of 0.5% saponin solution to the FACS tubes, incubate for 15 min in the dark at 4 &#176;C and then centrifuge at 4 &#176;C, 314 x g for 5 min.</li>
		<li>Stain the cells for intracellular markers (IL-17A and IFN&#947; or cytokines of choice) following the manufacturer&#39;s instructions. Wash the cells with 0.5% saponin solution and centrifuge at 4 &#176;C, 314 x g for 5 min.</li>
		<li>Wash again with FACS buffer and centrifuge at 4 &#176;C, 314 x g for 5 min.</li>
		<li>Re-suspend the cells in 300 &#181;l of FACS buffer.</li>
	</ol>
	</li>
	<li>Analyze results on a flow cytometer.</li>
</ol>
4. Flow Cytometry Analysis
<ol>
	<li>Visualize cells to be analyzed on forward (FSC) and side scatter (SCC) and gate to exclude debris.</li>
	<li>Select single cells with forward scatter area (FSC-A) vs. height (FSC-H) parameters.</li>
	<li>Gate cells that are live (as indicated by viability staining) and positive for the hematopoietic marker CD45+ for further analysis (Figure 2A and 2C).</li>
	<li>Continue analysis of specific markers as shown in Figure 2 and Figure 3.</li>
</ol>

<ol>
	<li>Aas, J. A., Paster, B. J., Stokes, L. N., Olsen, I., Dewhirst, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+The+Normal+Bacterial+Flora+Of+The+Oral+Cavity.">Defining The Normal Bacterial Flora Of The Oral Cavity.</a> J Clin Microbiol. 43, 5721-5732 (2005).</li><li>Belkaid, Y., Naik, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalized+And+Systemic+Control+Of+Tissue+Immunity+By+Commensals.">Compartmentalized And Systemic Control Of Tissue Immunity By Commensals.</a> Nat Immunol. 14, 646-653 (2013).</li><li>Darveau, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodontitis:+A+Polymicrobial+Disruption+Of+Host+Homeostasis.">Periodontitis: A Polymicrobial Disruption Of Host Homeostasis.</a> Nat Rev Microbiol. 8, 481-490 (2010).</li><li>Hajishengallis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomicrobial+Pathogenesis+Of+Periodontitis:+Keystones,+Pathobionts,+And+Host+Response.">Immunomicrobial Pathogenesis Of Periodontitis: Keystones, Pathobionts, And Host Response.</a> Trends Immunol. 35, 3-11 (2014).</li><li>Eke, P. 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=Prevalence+Of+Periodontitis+In+Adults+In+The+United+States:+2009+And+2010.">Prevalence Of Periodontitis In Adults In The United States: 2009 And 2010.</a> J Dent Res. 91, 914-920 (2012).</li><li>Moutsopoulos, N. M., Lionakis, M. S., Hajishengallis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inborn+Errors+In+Immunity:+Unique+Natural+Models+To+Dissect+Oral+Immunity.">Inborn Errors In Immunity: Unique Natural Models To Dissect Oral Immunity.</a> J Dent Res. , (2015).</li><li>Hajishengallis, G., Lamont, R. J., Graves, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Enduring+Importance+Of+Animal+Modelsin+Understanding+Periodontal+Disease.">The Enduring Importance Of Animal Modelsin Understanding Periodontal Disease.</a> Virulence. 6, 229-235 (2015).</li><li>Sharrow, S. O., Coligan, J. E., 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=Analysis+Of+Flow+Cytometry+Data.">Analysis Of Flow Cytometry Data.</a> Current Protocols In Immunology. , (2001).</li><li>Arizon, 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=Langerhans+Cells+Down-Regulate+Inflammation-Driven+Alveolar+Bone+Loss.">Langerhans Cells Down-Regulate Inflammation-Driven Alveolar Bone Loss.</a> Proc Natl Acad Sci U S A. 109, 7043-7048 (2012).</li><li>Moutsopoulos, N. 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=Defective+Neutrophil+Recruitment+In+Leukocyte+Adhesion+Deficiency+Type+I+Disease+Causes+Local+IL-17-Driven+Inflammatory+Bone+Loss.">Defective Neutrophil Recruitment In Leukocyte Adhesion Deficiency Type I Disease Causes Local IL-17-Driven Inflammatory Bone Loss.</a> Sci Transl Med. 6, 229-240 (2014).</li><li>Gaffen, S. L., Jain, R., Garg, A. V., Cua, 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=The+IL-23-IL-17+Immune+Axis:+From+Mechanisms+To+Therapeutic+Testing.">The IL-23-IL-17 Immune Axis: From Mechanisms To Therapeutic Testing.</a> Nat Rev Immunol. 14, 585-600 (2014).</li></ol>

Authors have nothing to disclose

The current technique successfully yields a large number of immune cells (from a single mouse) suitable, not only for phenotypic characterization, but also for functional studies ex vivo. Another group had previously published a protocol to isolate and characterize murine gingival immune cells and introduced the value of using multicolor flow cytometry in the study of periodontitis in animal models 9. Following considerable troubleshooting the key modification in this protocol was to include dissection of whole blocks of tissue in order to include both gingival collar areas and interdental gingiva (Figure 1). With gingival dissection alone, often full thickness flaps are not achieved and part of the gingiva is missed particularly in the interdental areas. Using this approach, numbers of immune cells harvested are significantly increased. This technique also allows for optimal isolation of gingival tissues both from maxilla and mandible.
Critical steps for this protocol include a conservative dissection of gingival areas and an efficient rate of dissection. Specifically, blocks of tissue should be dissected to include only minimal tissue around teeth (gingiva) and not buccal tissues. Blocks should be dissected quickly and tissues and cells should remain on ice to ensure optimal cell viability.
For purposes of troubleshooting it is important to note that low yield of cells can be related to inadequate time of incubation with collagenase, inappropriate concentrations of collagenase and/or inadequate dissection of the gingiva from the tissue blocks. On that note, dissection of gingiva using magnifying loupes (2X-6X) can be helpful. Low viability of cells can be related to tardiness in the procedure and to media, buffers and cells not remaining continuously on ice.
Limitations of this protocol, inherent to the type of tissue dissected is the size of the gingival tissues, compared to other anatomical barrier sites studied (ex. Skin or gastrointestinal tract) in which larger tissues are available.
Nonetheless, the technique presented here has allowed us to characterize the dominant cell populations in the LFA-/- model which spontaneously developed periodontitis. In LFA-/- mice we identified a dominant IL-17 cytokine signature during the development of periodontitis 10. Using multicolor flow cytometric analysis we were able to define the cellular sources of IL-17 11. In this disease setting IL-17 was produced by TCR&#947;&#948; T cells, CD4+ T cells and innate lymphoid cells (ILC) locally within the gingiva 10.
With the substantial number of cells isolated with this methodology and the ability to identify even small subpopulations, it will now be possible to develop a comprehensive understanding of the specialized immune cell network safeguarding gingival barrier integrity. This critical insight will allow us to evaluate immune responses in the gingival environment. By employing this technique we can now proceed to isolate specific constituents of the gingiva immune cell network by flow cytometric cell sorting, allowing additional analyses and characterizations to be undertaken.

Gingival tissues surround the human and murine dentition and are constantly exposed to the complex biofilm of the tooth 1. The immune cell network policing the gingival barrier is vital to maintaining tissue integrity, ensuring homeostasis with the local commensal microbes and, at the same time, providing effective immunity against pathogenic challenge 2. To achieve homeostasis, the immune system is carefully tailored to the gingival environment creating a highly-specialized immune cell network, yet little detail is known of the gingival immune cell populations and their role in maintaining tissue immunity 2.
When immune homeostasis is disrupted at the gingiva, either through increased host susceptibility and/or presence of dysbiotic microbial communities, an inflammatory condition, periodontitis arises 34. Periodontitis is a common inflammatory disease, leading to loss of tooth supporting structures. In its severe forms it is seen in approximately 10% of the general population 5. Dissecting the key factors involved in periodontitis susceptibility and progression has proven difficult 6. However, animal models have been extremely useful in understanding the mechanisms of periodontitis initiation and progression 7. Models can be employed to define the key cell populations and molecular mediators that are vital for maintaining immune homeostasis and drive development of periodontitis. Such insight will transform our understanding of gingiva-specific control of immune homeostasis and further our current understanding of disease pathogenesis.

<table border="0" cellpadding="0" cellspacing="0" width="914">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Fine Scissors</td>
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			<td style="width:188px;">6-304</td>
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			<td height="21" style="height:21px;width:304px;">Needles with regular bevel&#160;</td>
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			<td>27G, 12.7 mm length</td>
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			<td style="width:188px;">Life Technologies</td>
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			<td>Without Calcium and Magnesium</td>
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			<td style="width:188px;">12-167F</td>
			<td>Without L-glutamine</td>
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			<td height="21" style="height:21px;width:304px;">DNase I from bovine pancreas</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:188px;">DN25-1G</td>
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			<td height="20" style="height:20px;width:304px;">Collagenase type IV</td>
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			<td style="width:188px;">17104-019</td>
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			<td height="21" style="height:21px;width:304px;">Fetal Bovine Serum</td>
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			<td height="21" style="height:21px;width:304px;">Gentamicin 50 mg/ml</td>
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			<td height="42" style="height:42px;width:304px;">Pen Strep</td>
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			<td style="width:188px;">25030-081</td>
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			<td style="width:188px;">351-027-721EA</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:304px;">50 mL tubes</td>
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			<td style="width:188px;">352070</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:304px;">70 &#956;M Cell Strainers</td>
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			<td height="21" style="height:21px;width:304px;">Petri dishes</td>
			<td style="width:188px;">Corning</td>
			<td style="width:188px;">351029</td>
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			<td height="21" style="height:21px;width:304px;">5 mL FACS tubes</td>
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			<td style="width:188px;">352052</td>
			<td>Sterile</td>
		</tr>
		<tr height="21">
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			<td style="width:188px;">BD Biosciences</td>
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			<td>Contains brefeldin A solution</td>
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			<td height="21" style="height:21px;width:304px;">Phorbol 12-Myristate 13-Acetate (PMA)</td>
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			<td style="width:188px;">P8139</td>
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			<td height="21" style="height:21px;width:304px;">Ionomycin Calcium Salt</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:188px;">13909</td>
			<td></td>
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			<td height="21" style="height:21px;width:304px;">Saponin from quillaja bark</td>
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			<td style="width:188px;">S4521</td>
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			<td height="21" style="height:21px;">LIVE/DEAD Fixable Aqua Dead Cell Stain Kit</td>
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			<td>L34957</td>
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			<td style="width:188px;">56-0451-82</td>
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			<td style="width:188px;">48-0042-82</td>
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			<td height="21" style="height:21px;">Anti-Mouse TCR beta APC eFluor 780</td>
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			<td style="width:188px;">47-5961-82</td>
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			<td height="21" style="height:21px;">Anti-Mouse gamma delta TCR FITC</td>
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			<td style="width:188px;">11-5711-82</td>
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			<td height="21" style="height:21px;width:304px;">Anti-Mouse IL-17A APC</td>
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			<td style="width:188px;">12-7311-82</td>
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			<td>11-5931-82</td>
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			<td style="width:188px;">25-5941-82</td>
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			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD90.2 APC eFluor 780</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">47-0902-82</td>
			<td></td>
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			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD3e FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-0031-82</td>
			<td></td>
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			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD19 FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-0193-82</td>
			<td></td>
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			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD11b FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-0112-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD11c FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-0114-82</td>
			<td></td>
		</tr>
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			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-5961-82</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:304px;">Anti-Mouse Ly-6G FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-5931-82</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse Ly-6C FITC</td>
			<td style="width:188px;">BD Pharmingen</td>
			<td style="width:188px;">553104</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, characterization and functional examination of the gingival immune cell network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

To illustrate application of the protocol, we show representative results examining the immune cell network in the gingiva of mice with and without periodontitis (WT vs. LFA-/-, Figure 2A-C). Representative FACS plots show live CD45+ hematopoietic cells in the gingiva (Figure 2A, 2C). Isolation and processing of immune cells with this protocol yields sufficient cells to perform ex vivo stimulation and characterization of cytokine secretion patterns. Here we show IL-17 and IFN-&#947; staining in total CD45+ cells in the steady state (WT) and in mice exhibiting periodontitis (due to LFA deficiency) (Figure 2B-D). These data reveal the potential of this technique to capture cytokine profiles during tissue steady state (WT) and in the context of local periodontitis (LFA-/-). In mice susceptible to periodontitis we further characterized the gingival immune cell composition particularly of the T cell and Innate Lymphoid cell (ILC) compartments (Figure 3A-B). We were able to define the production of IL-17 and IFN-&#947; within the TCR&#946;+CD4+ T cell, TCR&#946;+CD8+ T cell, TCR&#947;&#948;+ T cell, NK and ILC compartments (Figure 3C).
<img alt="Figure 1" src="/files/ftp_upload/53736/53736fig1.jpg" /> 
Figure 1: Isolation of murine gingiva.&#160;Illustration demonstrating the boarders of dissection in the maxilla and the isolated gingival segment to be used for gingival tissue dissection. (B) Segment of murine maxilla (molar area) with outline for block dissection and isolated block. Hematoxylin and Eosin stain (H&#38;E) of a maxillary molar segment with gingival tissues outlined is shown in low (C) and higher magnification in (D). Original magnifications indicated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53736/53736fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53736/53736fig2.jpg" /> 
Figure 2: Representative FACS data for cytokine production from gingival cells.&#160;(A-C) FACS plot showing gate for live CD45+ cells in mouse gingival cell preparations following ex vivo re-stimulation with PMA and ionomycin in WT and LFA-/-. (B-D) Representative FACS plots showing IFN-&#947; and IL-17 staining in total CD45+ cells mouse from gingiva of mice with/without periodontitis (due to LFA-deficiency). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53736/53736fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53736/53736fig3.jpg" /> 
Figure 3: Cytokine production by immune populations in inflamed gingiva.&#160;(A) Representative FACS plot showing staining for TCR&#946; versus TCR&#947;&#948; gated on CD45+ cells. Gating on TCR&#946;+ cells, middle plot shows CD4 versus TCR&#946; staining (allows for analysis of TCR&#946;+CD4+ T cells, TCR&#946;+CD8+ T cells and TCR&#947;&#948;+ T cells). Gating on TCR&#946;- TCR&#947;&#948;- cells, right plot shows CD45 versus NK1.1 staining (allows for analysis of NK cells). (B) Representative FACS plot showing staining for CD90.2 gated on CD45+ lineage negative cells (CD3&#8722;CD19&#8722;NK1.1&#8722;CD11c&#8722;CD11b&#8722;Ly6G&#8722;Ly6C&#8722;TCR&#947;&#948;&#8722;TCR&#946;&#8722;) which identifies innate lymphoid cells. (C) Representative FACS plots showing IFN-&#947; and IL-17 staining in cell populations identified. Gingiva preparations are from 20 week old LFA-/- mice that develop periodontitis; data are representative of three independent experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53736/53736fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network at JoVE.com

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

isolation-characterization-functional-examination-gingival-immune

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.7K Views.  NIH. We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Video: Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Summary

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Chapters in this video

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Isolation of Functional Cardiac Immune Cells

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Isolation, Processing and Analysis of Murine Gingival Cells

Isolation of Adipose Tissue Immune Cells

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Isolation and Identification of Extravascular Immune Cells of the Heart