We thank Ivan Donati for providing of HA, Leonardo Amadio, Gabriella Zito (Department of Gynaecology of IRCCS &#34;Burlo Garofolo&#34;, Trieste, Italy) and Andrea Romano (Operative Clinical Unit of Anatomy and Pathological Histology, Cattinara Hospital, Trieste, Italy) for the tissue sample collection. We thank also Nicol&#242; Morosini for the help in the video preparation and Alex Coppola, the voice. This work was supported by grants from the Institute for Maternal and Child Health, IRCCS &#34;Burlo Garofolo&#34;, Trieste, Italy (RC20/16) and Fondazione Cassa di Risparmio Trieste to R.Bulla.

Tissue samples from patients were collected after informed consent following approval of the ethical considerations by the Institutional Board of the University Hospital of Trieste, Italy.
1. Tumor cell isolation and culture (Day 1)
<ol>
	<li>Isolate human mesothelioma cells from MPM solid specimens. Finely mince the tissue with a cutter to obtain fragments of about 2-3 mm2 in size and incubate in 5 mL of digestion solution composed of Hanks&#39; Balanced Salt Solution (HBSS) supplemented with 0.5 mM Ca2+/Mg2+, 0.5% trypsin and 50 &#181;g/mL DNase I, overnight at 4 &#176;C.</li>
	<li>Tumor cell isolation and culture (Day 2)
	<ol>
		<li>After overnight incubation, place the digested tissue for 30 min in a 37 &#176;C incubator with gentle shaking.</li>
		<li>Replace the digestion solution upon centrifugation (250 x g) with 3 mg/mL collagenase type 1 dissolved in 5 mL of Medium 199 with HBSS and further incubate for 30 min at 37 &#176;C with gentle shaking.</li>
		<li>Block the enzymatic reaction by adding 10% heat-inactivated fetal bovine serum (FBS). Resuspend the cells very carefully with a 5 mL pipet to ensure that most of the cells are released from the tissue. Then, filter the cell suspension through a 100 &#181;m pore filter.</li>
		<li>Seed the cells in a 12.5 cm2 flask and culture them at 37 &#176;C with human endothelial cells serum-free medium (HESF), with 10% FBS and supplemented with EGF (10 ng/mL), basic FGF (20 ng/mL), and 1% penicillin- streptomycin. 
		NOTE: Replace with fresh medium every 2-3 days.</li>
	</ol>
	</li>
</ol>
2. HA coating (Day 1)
<ol>
	<li>Resuspend high molecular weight HA (1.5 kDa) in double distilled water at the concentration of 1 mg/mL6.</li>
	<li>Dilute HA stock solution to 50 &#181;g/mL in carbonate/bicarbonate buffer (0.1 M, pH 9.6) with gentle&#160;pipetting.</li>
	<li>Coat the 96-well plate with 100 &#181;L of dilute HA stock solution per well overnight at 4 &#176;C. 
	NOTE: Hyaluronic acid was a kind gift from Professor Ivan Donati, Department of Life Sciences, University of Trieste7.</li>
</ol>
3. C1q coating (Day 2)
<ol>
	<li>After overnight incubation, vacuum aspirate the treated wells and wash the 96-well plate with 100 &#181;L of Dulbecco&#39;s PBS (dPBS) per well.</li>
	<li>Allow C1q (25 &#181;g/mL or different concentrations for dose response experiments) or BSA (as a negative control) to bind to plastic immobilized-HA by incubating these proteins at a concentration of 25 &#181;g/mL in 100 &#181;L of dPBS + 0.5% BSA and 0.7 mM Ca2+/Mg2+. Then incubate overnight at 4 &#176;C.</li>
	<li>Vacuum aspirate the wells and wash the 96-well plate with 100 &#181;L/well of dPBS.</li>
</ol>
4. Cell labeling with FAST DiI
<ol>
	<li>Resuspend 1 x 105 tumor cells in 500 &#181;L of dPBS containing 10 &#181;g/mL of the fluorescent dye FAST DiI. Incubate for 15 min at 37 &#176;C in a 5% v/v CO2 incubator for the labelling, mixing manually after 5 min intervals.
	<ol>
		<li>To remove excess FAST DiI, add 10 mL of dPBS, pipette gently up and down, and centrifuge at 250 x g for 7 min. Resuspend the cell pellet in 1 mL of human endothelial serum free medium containing 0.1% BSA (HESF + 0.1% BSA).</li>
	</ol>
	</li>
</ol>
5. Cell Adhesion on HA/C1q matrices (Day 1)
<ol>
	<li>Vacuum aspirate the wells coated with the different matrixes (wells were coated in step 3.2).</li>
	<li>Distribute 100 &#181;L of the labelled cell suspension to the coated wells and incubate the plate for 35 min at 37 &#176;C in 5% v/v CO2 incubator.</li>
	<li>Remove the non-adherent cells by aspirating the supernatant. Wash once with dPBS containing 0.5% BSA, 0.7 mM Ca2+ and 0.7 mM Mg2+.</li>
	<li>Lyse the adherent cells by adding 200 &#181;L/well of 10 mM Tris-HCl, pH 7.4 + 0.1% v/v SDS and immediately read the 96-well plate with a fluorescence reader (544 nm, emission 590 nm) using a calibration curve generated through the lysis of an increasing number of labelled cells.</li>
</ol>

<ol>
	<li>Zamboni, F., Vieira, S., Reis, R. L., Oliveira, J. M., Collins, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+hyaluronic+acid+in+immunoprotection+and+immunomodulation:+Chemistry,+processing+and+function.">The potential of hyaluronic acid in immunoprotection and immunomodulation: Chemistry, processing and function.</a> Progress in Materials Science. 97, 97-122 (2018).</li><li>Lorusso, G., Ruegg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tumor+microenvironment+and+its+contribution+to+tumor+evolution+toward+metastasis.">The tumor microenvironment and its contribution to tumor evolution toward metastasis.</a> Histochemistry and Cell Biology. 130 (6), 1091-1103 (2008).</li><li>Ricklin, D., Hajishengallis, G., Yang, K., Lambris, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement:+a+key+system+for+immune+surveillance+and+homeostasis.">Complement: a key system for immune surveillance and homeostasis.</a> Nature Immunology. 11 (9), 785-797 (2010).</li><li>Bulla, R., 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=C1q+acts+in+the+tumour+microenvironment+as+a+cancer-promoting+factor+independently+of+complement+activation.">C1q acts in the tumour microenvironment as a cancer-promoting factor independently of complement activation.</a> Nature Communications. 7, 10346 (2016).</li><li>Winslow, S., Leandersson, K., Edsjo, A., Larsson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+stromal+gene+signatures+in+breast+cancer.">Prognostic stromal gene signatures in breast cancer.</a> Breast Cancer Research. 17, 23 (2015).</li><li>Collins, M. N., Birkinshaw, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronic+acid+solutions:+A+processing+method+for+efficient+chemical+modification.">Hyaluronic acid solutions: A processing method for efficient chemical modification.</a> Journal of Applied Polymer Science. 130 (1), 145-152 (2013).</li><li>Itano, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+primary+structure,+complex+turnover+regulation+and+multiple+roles+of+hyaluronan.">Simple primary structure, complex turnover regulation and multiple roles of hyaluronan.</a> Journal of Biochemistry. 144 (2), 131-137 (2008).</li><li>Agostinis, 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=An+alternative+role+of+C1q+in+cell+migration+and+tissue+remodeling:+contribution+to+trophoblast+invasion+and+placental+development.">An alternative role of C1q in cell migration and tissue remodeling: contribution to trophoblast invasion and placental development.</a> Journal of Immunology. 185 (7), 4420-4429 (2010).</li><li>Agostinis, 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=Complement+Protein+C1q+Binds+to+Hyaluronic+Acid+in+the+Malignant+Pleural+Mesothelioma+Microenvironment+and+Promotes+Tumor+Growth.">Complement Protein C1q Binds to Hyaluronic Acid in the Malignant Pleural Mesothelioma Microenvironment and Promotes Tumor Growth.</a> Frontiers in Immunology. 8, 1559 (2017).</li><li>Bossi, F., 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=C1q+as+a+unique+player+in+angiogenesis+with+therapeutic+implication+in+wound+healing.">C1q as a unique player in angiogenesis with therapeutic implication in wound healing.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (11), 4209-4214 (2014).</li><li>Hosszu, K. 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=Cell+surface+expression+and+function+of+the+macromolecular+c1+complex+on+the+surface+of+human+monocytes.">Cell surface expression and function of the macromolecular c1 complex on the surface of human monocytes.</a> Frontiers in Immunology. 3, 38 (2012).</li><li>Laurent, T. C., Fraser, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan.">Hyaluronan.</a> Journal of the Federation of American Societies for Experimental Biology. 6 (7), 2397-2404 (1992).</li><li>Gaboriaud, 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=The+crystal+structure+of+the+globular+head+of+complement+protein+C1q+provides+a+basis+for+its+versatile+recognition+properties.">The crystal structure of the globular head of complement protein C1q provides a basis for its versatile recognition properties.</a> Journal of Biological Chemistry. 278 (47), 46974-46982 (2003).</li><li>Lam, J., Truong, N. F., Segura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+cell-matrix+interactions+in+hyaluronic+acid+hydrogel+scaffolds.">Design of cell-matrix interactions in hyaluronic acid hydrogel scaffolds.</a> Acta Biomaterialia. 10 (4), 1571-1580 (2014).</li></ol>

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

We describe&#160;an easy method to investigate how the complement component C1q, interacting with hyaluronic acid, is able to modulate the behavior of primary cells isolated from human tumor tissues. Both HA and C1q are abundantly present in the tissue microenvironment both under physiological and pathological conditions, participating to several cell biological processes. For instance, C1q has been shown to be present in the microenvironment of the placenta where it favors extravillous trophoblast invasion of the matern...

The tumor microenvironment (TME) influences cancer development and progression since it can provide a permissive niche for cell survival, growth and invasion. The identification of new key players in TME may be useful for the discovery of new molecular tools for target therapy. TME includes a complex and dynamic network of non-malignant cells, such as endothelial cells, fibroblasts and cells of the immune system, embedded in the surrounding extracellular matrix (ECM) components including collagens, laminins, fibronectins, proteoglycans and hyaluronans. Both tumor and non-tumor cells synthesize and secrete ECM components together with cytokines, chemokines, growth factors and inflammatory and matrix remodeling enzymes that overall alter the physical, chemical and signaling properties of TME. Among these constituents, hyaluronic acid (HA) has emerged to exert a crucial role in tumor biology. Despite its simple chemical composition, HA, together with its HA-binding molecules (hyaladherins), can modulate angiogenesis, immune system responsiveness and ECM remodeling in a size and concentration dependent manner1.
The complement (C) system is also part of the local TME, which has recently received increasing attention. The C system encompasses a set of soluble and membrane-bound proteins involved in the first line of defense against non-self-cells, unwanted host elements and pathogens. Functionally, the C links the two-effector arms of innate and adaptive systems to promote either direct cell killing or mounting of an inflammatory response2. C activation can suppress tumor growth, by destroying cancer cells or inhibiting their outgrowth, but it has become increasingly clear that it can possess a tumor-promoting activity by sustaining chronic inflammation, promoting the establishment of an immunosuppressive milieu, inducing angiogenesis, and activating cancer-related signaling pathways3. In this context, C1q, the first recognition molecule of the classical pathway of the C system has emerged to exert important functions in the tumor microenvironment independently of C activation4. C1q has been shown to be expressed locally by a range of human malignant tumors, where it can favor cancer cell adhesion, migration and proliferation in addition to angiogenesis and metastasis5. Interestingly C1q interacts with a major constituent of the ECM such as HA.
We developed a technique to isolate the primary cancer cells from the tumor mass. Furthermore, we created the matrix, which can stimulate tumor microenvironment, particularly the interaction between C1q and high molecular weight hyaluronic acid. C1q bound to HA was able to induce adhesion of the tumor cells.

<table border="0" cellpadding="0" cellspacing="0" style="width:1447px;" width="1445">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:330px;">100&#8201;&#181;m pore filter</td>
			<td style="width:311px;">BD Falcon</td>
			<td style="width:131px;">352360</td>
			<td style="width:676px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:330px;">Amphotericin B solution (fungizone)</td>
			<td style="width:311px;">Sigma-Aldrich&#160;</td>
			<td style="width:131px;">1397-89-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:330px;">basic FGF</td>
			<td style="width:311px;">Immunological Sciences</td>
			<td style="width:131px;">GRF-15595</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:330px;">Calcium Chloride</td>
			<td style="width:311px;">Sigma-Aldrich&#160;</td>
			<td style="width:131px;">C-4901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase type I</td>
			<td>Worthington Biochemical Corporation, DBA</td>
			<td style="width:131px;">MX1D12644</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:330px;">D-Glucose</td>
			<td style="width:311px;">Sigma-Aldrich&#160;</td>
			<td style="width:131px;">50-99-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:330px;">DNase I&#160;</td>
			<td style="width:311px;">Roche</td>
			<td style="width:131px;">10 104 159 001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:330px;">EDTA</td>
			<td style="width:311px;">Sigma-Aldrich&#160;</td>
			<td style="width:131px;">60-00-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:330px;">EGF</td>
			<td style="width:311px;">Immunological Sciences</td>
			<td style="width:131px;">GRF-10544</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:330px;">FAST DiI</td>
			<td style="width:311px;">Molecular probes, Invitrogen, Thermo Fisher Scientific</td>
			<td style="width:131px;">D7756</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td style="width:311px;">Gibco,&#160; Thermo Fisher Scientific</td>
			<td style="width:131px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:330px;">Fibronectin</td>
			<td style="width:311px;">Roche</td>
			<td style="width:131px;">11051407001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:330px;">Flask for cell culture</td>
			<td style="width:311px;">Corning</td>
			<td style="width:131px;">430639</td>
			<td>Sterile, vented</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:330px;">Gentamicin solution</td>
			<td style="width:311px;">Sigma-Aldrich&#160;</td>
			<td style="width:131px;">G1397-10ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hank&#8217;s Balanced Salt Solution (HBBS)&#160;</td>
			<td style="width:311px;">Sigma-Aldrich&#160;</td>
			<td style="width:131px;">H6648</td>
			<td>Supplemented with EDTA, Glucose, penicillin-streptamicin, gentamicin and fungizone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:330px;">High molecular weight hyaluronic acid</td>
			<td style="width:311px;">Kind gift by Prof. Ivan Donati</td>
			<td style="width:131px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:330px;">Human endothelial serum free medium&#160;</td>
			<td style="width:311px;">Gibco,&#160; Thermo Fisher Scientific</td>
			<td style="width:131px;">11111-044</td>
			<td>Supplemented with EGF (5&#8201;ng/mL), basic FGF (10&#8201;ng/mL), and&#160; 1% penicillin&#8211;streptomycin (Sigma-Aldrich)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:330px;">Magnesium Chloride</td>
			<td style="width:311px;">Carlo Erba</td>
			<td style="width:131px;">13446-18-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Medium 199 with Hank&#8217;s salt</td>
			<td style="width:311px;">Sigma-Aldrich&#160;</td>
			<td style="width:131px;">M7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:330px;">Penicillin-Streptomycin</td>
			<td style="width:311px;">Sigma-Aldrich&#160;</td>
			<td style="width:131px;">P0781</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:330px;">Time-lapse microscopy&#160;</td>
			<td style="width:311px;">Nikon&#160;</td>
			<td style="width:131px;">Imaging System BioStation IM-Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Titertek Multiskan ELISA Reader</td>
			<td>Flow Labs</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin</td>
			<td style="width:311px;">Sigma-Aldrich&#160;</td>
			<td style="width:131px;">T4674</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of the interplay between the complement protein c1q and hyaluronic acid in promoting cell adhesion

It has been increasingly demonstrated that the tumor microenvironment plays an active role in neoplasia growth and metastasis. Through different pathways, tumor cells can efficiently recruit stromal, immune and endothelial cells by secreting stimulatory factors, chemokines and cytokines. In turn, these cells can alter the signaling properties of the microenvironment by releasing growth-promoting signals, metabolites and extracellular matrix components to sustain high proliferation and metastatic competence. In this context, we identify that the complement component C1q, highly expressed locally by a range of human malignant tumors, upon interacting with the extracellular matrix hyaluronic acid, strongly affects the behavior of primary cells isolated from human tumor specimens. Here, we describe&#160;a method to test how C1q bound to hyaluronic acid (HA) impacts tumor cell adhesion, underlying the fact that the biological properties of key components of the extracellular matrix (in this case HA) can be shaped by bioactive signals toward tumor progression.

HA is a negatively charged high-molecular-weight polysaccharide, which is made up of repeating (&#946;,1-4)-D-glucuronic acid-(&#946;,1-3)-N-acetyl-D-glucosamine disaccharide units (Figure 1B)7. The occurrence of the binding of HA on the 96-well plate as well as the efficiency of its immobilization were tested taking advantage of biotinylated HA (bio-HA). Different concentrations of Bio-HA, ranging from 10 &#181;g/mL to 1 mg/mL, were r...

Watch this Scientific Journal Video about Evaluation of the Interplay Between the Complement Protein C1q and Hyaluronic Acid in Promoting Cell Adhesion at JoVE.com

Evaluation of the Interplay Between the Complement Protein C1q and Hyaluronic Acid in Promoting Cell Adhesion

The complement component C1q is a pro-inflammatory molecule highly expressed in the tissue microenvironment that can interact with the extracellular matrix. Here, we describe&#160;a method to test how C1q bound to hyaluronic acid impacts cell adhesion.

It has been increasingly demonstrated that the tumor microenvironment plays an active role in neoplasia growth and metastasis. Through different pathways, ...

evaluation-interplay-between-complement-protein-c1q-hyaluronic-acid

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Immunology and Infection

5.9K Views.  University of Trieste. The complement component C1q is a pro-inflammatory molecule highly expressed in the tissue microenvironment that can interact with the extracellular matrix. Here, we describe a method to test how C1q bound to hyaluronic acid impacts cell adhesion.

Video: Evaluation of the Interplay Between the Complement Protein C1q and Hyaluronic Acid in Promoting Cell Adhesion

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Measuring the 50% Haemolytic Complement (CH50) Activity of Serum

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual complement components can be quantified however this does not provide any information as to the activity of the pathway. The CH50 is a screening assay for the activation of the classical complement pathway (Fig 1) and it is sensitive to the reduction, absence and/or inactivity of any component of the pathway. The CH50 tests the functional capability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (haemolysin). When antibody-coated SRBC are incubated with test serum, the classical pathway of complement is activated and haemolysis results. If a complement component is absent, the CH50 level will be zero; if one or more components of the classical pathway are decreased, the CH50 will be decreased. A fixed volume of optimally sensitised SRBC is added to each serum dilution. After incubation, the mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of the haemoglobin released into the supernatant at 540nm. The amount of complement activity is determined by examining the capacity of various dilutions of test serum to lyse antibody coated SRBC. This video outlines the experimental steps involved in analysing the level of complement activity of the classical complement pathway.

Measuring the 50% Haemolytic Complement CH50 Activity of Serum

The classical pathway is activated by antibody and culminates in target cell lysis. The CH50 assay provides a measure of the complement activity of a serum sample. This video demonstrates the steps involved in determining the CH50 of a serum sample, the calculations and interpreting of results.

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-&#945;4&#946;7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn&#39;s disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.
The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.
Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Renal Subcapsular Transplantation of 2'-Deoxyguanosine-Treated Murine Embryonic Thymus in Nude Mice

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation is an important method for investigating thymic epithelial cell function and T cells maturation in vivo. Here we will describe the experimental methods used within our laboratory to transplant 2&#8217;-deoxyguanosine (to deplete donor&#8217;s lymphocytes) treated embryonic thymus into the renal capsule of an athymic nude mouse. This method is both simple and efficient and does not require special skills or devices. The results obtained via this simple method showed that transplanted thymus can effectively support the recipient&#8217;s T cells production. Additionally, several key points with regards to the protocol will be further elucidated.

We provide a simple and efficient method to transplant 2&#8217;-deoxyguanosine treated E18.5 thymus into the renal capsule of a nude mouse. This method should aide in the study of both thymic epithelial cells function and T cells maturation.

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation ...

Evaluation of the Interplay Between the Complement Protein C1q and Hyaluronic Acid in Promoting Cell Adhesion

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