Name: isolation, characterization and functional examination of the gingival immune cell network
Uploaded: 2016-02-16T15:00:00
Description: Watch this Scientific Journal Video about Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network at JoVE.com

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...

<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>

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 dissectio...

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>
			<td style="width:188px;">Fine science tools</td>
			<td style="width:188px;">14058-11</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Scalpel Handle #3</td>
			<td style="width:188px;">Fine science tools</td>
			<td style="width:188px;">10003-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Scalpel Blades #10</td>
			<td style="width:188px;">Fine science tools</td>
			<td style="width:188px;">10010-00</td>
			<td>Sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Splinter Forceps</td>
			<td style="width:188px;">Integra Miltex</td>
			<td style="width:188px;">6-304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Needles with regular bevel&#160;</td>
			<td style="width:188px;">BD Medical</td>
			<td style="width:188px;">305109</td>
			<td>27G, 12.7 mm length</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Monoject syringes</td>
			<td style="width:188px;">Covidien</td>
			<td style="width:188px;">8881513934</td>
			<td>Luer-lock tip, 3mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">PBS, pH 7.4</td>
			<td style="width:188px;">Life Technologies</td>
			<td style="width:188px;">10010-049</td>
			<td>Without Calcium and Magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">RPMI 1640</td>
			<td style="width:188px;">Lonza</td>
			<td style="width:188px;">12-167F</td>
			<td>Without L-glutamine</td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:304px;">Collagenase type IV</td>
			<td style="width:188px;">Gibco (by Life technologies)</td>
			<td style="width:188px;">17104-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Fetal Bovine Serum</td>
			<td style="width:188px;">Gemini Bio-products</td>
			<td style="width:188px;">100-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Gentamicin 50 mg/ml</td>
			<td style="width:188px;">Quality biological</td>
			<td style="width:188px;">120-098-661EA</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:304px;">Pen Strep</td>
			<td style="width:188px;">Gibco (by Life technologies)</td>
			<td style="width:188px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:304px;">L-Glutamine</td>
			<td style="width:188px;">Gibco (by Life technologies)</td>
			<td style="width:188px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">0.5M EDTA pH 8.0</td>
			<td style="width:188px;">Quality biological</td>
			<td style="width:188px;">351-027-721EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">50 mL tubes</td>
			<td style="width:188px;">Corning</td>
			<td style="width:188px;">352070</td>
			<td>Polypropylene, sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">70 &#956;M Cell Strainers</td>
			<td style="width:188px;">Corning</td>
			<td style="width:188px;">352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Petri dishes</td>
			<td style="width:188px;">Corning</td>
			<td style="width:188px;">351029</td>
			<td>Sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">5 mL FACS tubes</td>
			<td style="width:188px;">Corning</td>
			<td style="width:188px;">352052</td>
			<td>Sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD GolgiPlug</td>
			<td style="width:188px;">BD Biosciences</td>
			<td style="width:188px;">555029</td>
			<td>Contains brefeldin A solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Phorbol 12-Myristate 13-Acetate (PMA)</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:188px;">P8139</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Saponin from quillaja bark</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:188px;">S4521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD Fixable Aqua Dead Cell Stain Kit</td>
			<td style="width:188px;">Life Technologies</td>
			<td>L34957</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD45 Alexa Fluor 700</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">56-0451-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD4 eFluor 450</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">48-0042-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Mouse TCR beta APC eFluor 780</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">47-5961-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Mouse gamma delta TCR FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-5711-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse IL-17A APC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">12-7311-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse IFN-&#947; PE</td>
			<td style="width:188px;">eBioscience</td>
			<td>11-5931-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse NK1.1 PE-Cy7</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">25-5941-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse TCR beta FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-5961-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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 stimu...

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 ...

The overall goal of this method is to isolate gingival immune cells for the in-depth characterization of the immune responses related to oral immunity and periodontitis. This method can help answer key questions in the field of oral immunity, such as what cellular factors are implicated in the pathogenesis of periodontitis. The main advantages of this technique are that it allows the evaluation of the oral immune cell network and the isolation of enough viable cells for downstream in-vitro investigations.As a first step of this protocol, begin by placing pins to immobilize the body of the mouse and open the thoracic and abdominal cavities to expose the heart and internal organs. Next, make an incision in the vena cava, and use a 3-milliliter syringe, equipped with a 27-gauge needle, to profuse 3 milliliters of PBS via the left ventricle. When all of the blood has been flushed, pin the head of the mouse onto a dissecting pad, with the stomach facing up.Then use scissors to cut each commissure of the lip towards the neck, separating the skin that covers the mandible and neck region, and exposing the underlying tissues and muscles. Cut between the lower incisors to separate both sides of the mandible. Immobilize each side of the separated mandible with 25-gauge needles to access the oral cavity.After identifying the palate, use a number 10 surgical blade to make vertical incisions just anterior to the first molar, and posterior to the third molar. Then dissect the tissue away from the nasal cavity, and also dissect the blocks from the mandible. Then eliminate the excess tissue from the border of the gingiva and transfer the maxillary and mandibular blocks into a 50-milliliter conical tube containing 5 milliliters of collagenase and DNase on ice.Digest the tissue in a shaker incubator at 37 degrees Celsius for one hour, adding 50 microliters of 0.5 molar EDTA during the last five minutes. Then add 5 milliliters of cold DNase medium to the tissues, and gently mix the tube contents by swirling. Next, transfer the four blocks of tissue into a petri dish.And cover them with 500 microliters of fresh DNase medium. Use a scalpel to remove the gingiva from each block of tissue. And transfer the entire contents of the dish into a 70 micron cell strainer on top of a 50-milliliter tube.Wash the petri dish and strainer with 3 to 5 milliliters of fresh DNase medium. And then use the plunger of a sterile, 3-milliliter syringe to mash the gingival tissue against the mesh of the strainer. Rinse the strainer with 30 to 35 millilters of cold DNase medium, and centrifuge the filtrate.Then resuspend the palate in 1 milliliter of complete medium, and determine the number of viable cells. The isolation and processing of immune cells as just demonstrated, yields a sufficient number of cells to perfom an ex-vivo stimulation and characterization of the gingival cell cytokine secretion patterns from both wild-type, as well as periodontitis-susceptible animals. Indeed, the murine gingival immune cell composition can be analyzed as demonstrated in these density plots evaluating the t and natural killer cell compartments in periodontitis-susceptible LFA knockout mice.Using the appropriate gating strategies, the intracellular cytokine production of TCR beta CD4 positive, TCR beta CD8 positive, and TCR gamma delta positive t cells, as well as NK, and innate lymphoid cells, can also be determined. Once mastered, the cells can be isolated in three hours for immunostaining if the technique is performed properly. While attempting this procedure, it's important to remember to complete all of the steps efficiently and quickly, keeping the cells on ice whenever they are not being manipulated.Following this procedure, other methods, like cell sorting, can be performed to answer additional questions related to the function of the specific cell subsets.

Research

JoVE Journal

Immunology and Infection

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, ...

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 ...

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 ...

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 ...

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 ...

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

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. ...

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 ...

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 ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...