This work was supported by the Fondation Laurette Fugain (LF-2015-15) (B.S.M.) and ANR JCJC grants (ANR-17-CE15-0012) (B.S.M.).

All methods described here have been reviewed and approved by the French organizations CoRC (Comit&#233; de Recherche Clinique), CPP (Comit&#233; de Protection des Personnes), and CNIL (Commission Nationale Informatique et Libert&#233;).
1. Staining Neutrophils in Histopathology Tissue with Fluorescently Labelled MUB40
<ol>
	<li>To obtain the tissue for histopathology, fix tissue/biopsy sections in a suitable volume of 4% paraformaldehyde (PFA) for 30 min up to 4 h depending on the thickness of the sample. Place the samples in a 4 &#176;C refrigerator during fixation. 
	Note: Alternative fixation methods/timings can be substituted based on the user&#39;s unique needs.
	<ol>
		<li>Remove the PFA, add 16% sucrose solution with enough volume to completely cover the sample, and place the sample at 4 &#176;C for 4 h up to overnight.</li>
		<li>Remove 16% sucrose solution and add 30% sucrose solution at a large enough volume to completely cover the sample, and place it at 4 &#176;C for 4 h up to overnight.</li>
		<li>Remove the 30% sucrose solution and blot off excess solution from the tissue with a paper towel. Place the tissue section in optimal cutting temperature (OCT) media and snap-freeze in 2-methylbutane cooled in a dry ice-ethanol bath (-72 &#176;C). Once frozen solid (after ~2 min), remove blocks from the 2-methylbutane and place them on dry ice until all blocks are finished and ready for long-term storage. Store the frozen blocks at -80 &#176;C for long-term storage.</li>
	</ol>
	</li>
	<li>The night before tissue blocks are to be cut, remove them from the -80 &#176;C freezer and place them at -20 &#176;C overnight to equilibrate.
	<ol>
		<li>Using a cryostat, cut OCT-embedded tissue into 10- to 30 &#956;m-thick slices and adsorb to high-quality glass slides. 
		Note: Thicker or thinner slices can be used depending on the experimental requirements.</li>
		<li>With a wax pen or other hydrophobic method, draw a box around the tissue slice on the glass slide and let dry.</li>
		<li>Prepare the staining solution by performing a 1:1,000 dilution of MUB40-Cy5 (or other fluorescent MUB40version, 1 mg/mL stock solution) in 0.1% saponin-PBS solution. The optimal final MUB40 concentration should be 1 &#956;g/mL. 
		Note: Lower final concentrations may be used (0.1-0.01 &#956;g/mL) but may lead to lower signal intensity. 
		CAUTION: Saponin powder can cause breathing irritation. Make sure to weigh saponin in a cabinet or protected area. Alternative permeabilization methods can be used such as Triton. Add additional markers at manufacturer-recommended concentrations (e.g., 4&#8242;,6-diamidino-2-phenylindole (DAPI), phalloidin, fluorescent antibodies).</li>
		<li>Gently dip the glass slide into a solution of 0.1% saponin-PBS three times.</li>
		<li>Carefully blot away excess liquid around the tissue section and add enough staining solution to completely cover the tissue section. Place the slide into a humidity chamber to avoid evaporation and incubate it for 1 h at room temperature. Keep it protected from light.</li>
		<li>Gently dip the slide into 0.1% saponin-PBS solution 3x. Then, gently dip the slide into PBS solution 3x. Finally, gently dip the slide into distilled H2O 3x. Carefully dry excess liquid from the slide.</li>
		<li>Add a small amount (~20 &#956;L) of mounting medium (20 mM Tris pH 8.0, 0.5% N-propyl gallate, 90% glycerol) next to the tissue slice and gently lay down a glass coverslip on top of the glass slide. Gently and evenly press the coverslip onto the tissue section and, using a paper towel, carefully remove any mounting media that extrudes around the edges of the coverslip.</li>
		<li>Place the mounted slide in a dark cabinet for 24 h to allow the mounting media to solidify. Alternatively, place the mounted slide at 37 &#176;C for 1 h.</li>
	</ol>
	</li>
	<li>Image the tissue slices on a fluorescent microscope according to the desired acquisition method.</li>
</ol>
2. Visualization of Neutrophil Activation with Retro-Inverso-MUB40-Cy5 Peptide
<ol>
	<li>Obtain human neutrophils using the following protocol. Prepare the following solutions and place them in an anoxic cabinet overnight. Exposure to oxygen will begin to activate neutrophils and further induce cell death16,17. 
	Note: Neutrophils may be alternatively purified &#34;on the bench&#34; for MUB40-labeling experiments; however, be aware that oxygen exposure will begin to affect neutrophil activation.
	<ol>
		<li>Prepare the following solution: 
		Solution 1 [sodium chloride (NaCl) 0.9%]: add 4.5 g of NaCl to 500 mL of H2O. Filter the solution. 
		Solution 2 (dextran 6%): add 6 g of dextran in NaCl 0.9% solution to 100 mL. 
		Solution 3 (washing buffer): dissolve ethylenediaminetetraacetic acid (EDTA) in PBS pH 7.2 to a final EDTA concentration of 2 mM. Immediately prior to use, dissolve bovine serum albumin (BSA) in the PBS/EDTA solution such that the final BSA concentration is 10% w/v.</li>
		<li>Centrifuge blood collection tubes at 650 x g for 20 min without breaks.</li>
		<li>Without disrupting separated blood, transfer the blood collection tubes to an anoxic cabinet and collect plasma fractions (top) in a 50 mL conical tube.</li>
		<li>Remove the plasma-containing tube from the anoxic cabinet and centrifuge at 2,900 x g for 20 min with breaks to pellet the platelets.</li>
		<li>Without disrupting the pelleted platelets, gently transfer the plasma tube back into the anoxic cabinet and pipet the platelet poor plasma into a fresh 50 mL tube (hereafter called &#34;plasma&#34;).</li>
	</ol>
	</li>
	<li>Separation of red blood cells from leukocytes
	<ol>
		<li>Using the tubes containing the red blood cells (RBCs) from step 2.1.3., combine the RBCs into a conical 50 mL tube (5 blood collection tube contents per 50 mL tube).</li>
		<li>Add NaCl 0.9% until the total volume reaches 44 mL. Add 6 mL of dextran 6% (last).</li>
		<li>Gently mix the blood/dextran mixture by inverting the tube 10-20 times. Let the tube sediment for at least 30 min.</li>
		<li>While sedimentation is occurring, prepare a Percoll gradient by adding 4.2 mL of Percoll solution to 5.8 mL of plasma in a 15 mL conical tube, and mix well by inverting the tube.</li>
		<li>Collect the top fraction of the dextran while trying to avoid pipetting red blood cells.</li>
		<li>Tighten the screw cap, remove the dextran tube from the anoxic chamber, and centrifuge the tube at 300 x g for 10 min with breaks.</li>
		<li>Carefully place the tube back into the anoxic chamber without disturbing the pelleted cells, and remove the liquid via pipetting.</li>
		<li>Gently re-suspend the cell pellet in 1 mL of plasma and very slowly add to the top of the Percoll solution. Be careful not to induce mixing of the layers when pipetting the cells on top.</li>
		<li>Carefully remove the Percoll solution tube from the anoxic chamber (avoiding mixing), and centrifuge at 800 x g for 20 min without breaks. Peripheral blood mononuclear cells (PBMCs) will remain on the Percoll solution surface, and neutrophils will pellet with the RBCs.</li>
	</ol>
	</li>
	<li>Removal of contaminating RBCs
	<ol>
		<li>Prepare 14 mL of washing buffer solution by adding 700 &#956;L PBS + 10% BSA to 14 mL of washing buffer.</li>
		<li>Place the Percoll solution tube back into the anoxic chamber. Remove the Percoll and PBMCs via pipetting and re-suspend the pelleted cells in 1 mL of washing buffer solution. The pellet to be re-suspended will have a red color due to contaminating RBCs.</li>
		<li>Add 200 &#956;L of anti-CD235a (glycophorin) magnetic beads to the re-suspended cells, gently mix, and incubate for a minimum of 15 min. This step loads the contaminating RBCs with magnetic beads so that they can be removed.</li>
		<li>Wash the separation column with 2 mL of washing buffer 3x.</li>
		<li>While holding a 15 mL conical tube under the separation column, slowly pipette the neutrophil/RBC mixture to the top of the column. Collect the cells in the 15 mL conical tube as they flow through. The flow-through should be cloudy and lose its red color due to the RBCs remaining bound to the column.</li>
		<li>Add 2 mL of washing buffer to the top of the column and collect in the same 15 mL conical tube. By the end of the 2 mL wash, the flow-through should be clear, signifying that all the neutrophils have been collected.</li>
		<li>Gently mix the tube to ensure even distribution of neutrophils and collect 10 &#956;L for cell counting. Note the final volume for cell counting purposes. Enumerate the total number of cells with any standard cell counting method.</li>
		<li>Remove the neutrophil tube from the anoxic chamber, and centrifuge the neutrophils at 300 x g for 20 min with breaks.</li>
		<li>Place sedimented neutrophils back into the anoxic chamber and remove the washing buffer via pipetting. Re-suspend the neutrophil pellet in plasma with a maximum cell density of 107&#160;PMN/mL.</li>
	</ol>
	</li>
	<li>Enumeration of purified neutrophils and visualization using MUB40
	<ol>
		<li>Add 1 &#956;L of RI-MUB40-Cy5 (1mg/mL) to 1 mL of Rosewell Park Memorial Institute (RPMI) 1640 medium without phenol red. Mix by pipetting well.</li>
		<li>For the purposes of this protocol, the result has been outlined as if a potent neutrophil activating reagent, N-formylmethionyl-leucyl-phenylalanine (FMLP), is added. Add 1 &#956;m of FMLP to the RPMI/RI-MUB40-Cy5 tube and mix via pipetting up and down. 
		NOTE: Each experimental procedure will be different from this point forward, depending on the questions being addressed.</li>
		<li>Transfer the 1 mL RPMI/RI-MUB40-Cy5/FMLP mixture to a glass bottom microscopy dish. To this dish, add a volume of purified cells corresponding to 106&#160;total neutrophils. Mix by gently pipetting.</li>
		<li>Place the dish into an inverted fluorescent microscope and start image acquisition. For example, it is recommended to acquire baseline images prior to the addition of a neutrophil-activating reagent such as FMLP or bacteria. In this example, start the acquisition of RPMI/RI-MUB40-Cy5/neutrophils, and at a later timepoint, pipette the neutrophil-activating reagent into the glass dish and continue image acquisition. 
		NOTE: Modifications to these steps can be made based on scientific needs.</li>
		<li>Process acquired images/time-lapse movies as pertains to the specific microscope used.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Nathan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+and+immunity:+challenges+and+opportunities.">Neutrophils and immunity: challenges and opportunities.</a> Nature Reviews Immunology. 6 (3), 173-182 (2006).</li><li>Nicol&#225;s-&#193;vila, J. &. #. 1. 9. 3. ;., Adrover, J. M., Hidalgo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+in+Homeostasis,+Immunity,+and+Cancer.">Neutrophils in Homeostasis, Immunity, and Cancer.</a> Immunity. 46 (1), 15-28 (2017).</li><li>Coffelt, S. B., Wellenstein, M. D., de Visser, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+in+cancer:+neutral+no+more.">Neutrophils in cancer: neutral no more.</a> Nature Reviews Cancer. 16 (7), 431-446 (2016).</li><li>Wright, H. L., Moots, R. J., Bucknall, R. C., Edwards, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+function+in+inflammation+and+inflammatory+diseases.">Neutrophil function in inflammation and inflammatory diseases.</a> Rheumatology. 49 (9), 1618-1631 (2010).</li><li>Glennon-Alty, L., Hackett, A. P., Chapman, E. A., Wright, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+and+redox+stress+in+the+pathogenesis+of+autoimmune+disease.">Neutrophils and redox stress in the pathogenesis of autoimmune disease.</a> Free Radical Biology &amp; Medicine. , (2018).</li><li>Borregaard, N., Cowland, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granules+of+the+human+neutrophilic+polymorphonuclear+leukocyte.">Granules of the human neutrophilic polymorphonuclear leukocyte.</a> Blood. 89 (10), 3503-3521 (1997).</li><li>Borregaard, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils,+from+marrow+to+microbes.">Neutrophils, from marrow to microbes.</a> Immunity. 33 (5), 657-670 (2010).</li><li>Rose, S., Misharin, A., Perlman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+Ly6C/Ly6G-based+strategy+to+analyze+the+mouse+splenic+myeloid+compartment.">A novel Ly6C/Ly6G-based strategy to analyze the mouse splenic myeloid compartment.</a> Cytometry Part A: The Journal of the International Society for Analytical Cytology. 81 (4), 343-350 (2012).</li><li>Shi, C., Hohl, T. M., Leiner, I., Equinda, M. J., Fan, X., Pamer, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ly6G++neutrophils+are+dispensable+for+defense+against+systemic+Listeria+monocytogenes+infection.">Ly6G+ neutrophils are dispensable for defense against systemic Listeria monocytogenes infection.</a> Journal of Immunology. 187 (10), 5293-5298 (2011).</li><li>Albanesi, M., Mancardi, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+mediate+antibody-induced+antitumor+effects+in+mice.">Neutrophils mediate antibody-induced antitumor effects in mice.</a> Blood. 122 (18), 3160-3164 (2013).</li><li>Anderson, M. C., Chaze, T., 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=MUB40+Binds+to+Lactoferrin+and+Stands+as+a+Specific+Neutrophil+Marker.">MUB40 Binds to Lactoferrin and Stands as a Specific Neutrophil Marker.</a> Cell Chemical biology. 25 (4), 483-493 (2018).</li><li>Co&#239;c, Y. -. M., Baleux, 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=Design+of+a+specific+colonic+mucus+marker+using+a+human+commensal+bacterium+cell+surface+domain.">Design of a specific colonic mucus marker using a human commensal bacterium cell surface domain.</a> Journal of Biological Chemistry. 287 (19), 15916-15922 (2012).</li><li>Roos, S., Jonsson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-molecular-mass+cell-surface+protein+from+Lactobacillus+reuteri+1063+adheres+to+mucus+components.">A high-molecular-mass cell-surface protein from Lactobacillus reuteri 1063 adheres to mucus components.</a> Microbiology. 148, 433-442 (2002).</li><li>Boekhorst, J., Helmer, Q., Kleerebezem, M., Siezen, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+proteins+with+a+mucus-binding+domain+found+exclusively+in+lactic+acid+bacteria.">Comparative analysis of proteins with a mucus-binding domain found exclusively in lactic acid bacteria.</a> Microbiology. 152, 273-280 (2006).</li><li>Mancardi, D. A., J&#246;nsson, 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=Cutting+Edge:+The+murine+high-affinity+IgG+receptor+Fc&#947;RIV+is+sufficient+for+autoantibody-induced+arthritis.">Cutting Edge: The murine high-affinity IgG receptor Fc&#947;RIV is sufficient for autoantibody-induced arthritis.</a> Journal of Immunology. 186 (4), 1899-1903 (2011).</li><li>Walmsley, S. R., Print, 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=Hypoxia-induced+neutrophil+survival+is+mediated+by+HIF-1alpha-dependent+NF-kappaB+activity.">Hypoxia-induced neutrophil survival is mediated by HIF-1alpha-dependent NF-kappaB activity.</a> Journal of Experimental Medicine. 201 (1), 105-115 (2005).</li><li>Monceaux, V., Chiche-Lapierre, 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=Anoxia+and+glucose+supplementation+preserve+neutrophil+viability+and+function.">Anoxia and glucose supplementation preserve neutrophil viability and function.</a> Blood. 128 (7), 993-1002 (2016).</li></ol>

B.S.M. is listed as an inventor on MUB40&#160;patents:
MUB40: European Patent EP11290403.2, 09/09/2011.
RI-MUB40: European Patent EP no. 17306746.3, 11/12/17.

Here, two assays are described in which the MUB40&#160;peptide is used to study neutrophil inflammation and activation. We show how MUB40&#160;can reveal neutrophils present in histopathology sections or show their activation state using live purified neutrophils. The critical steps for using MUB40 as a staining reagent are the same as with any other fluorescent detection method. Care must be taken to ensure compatibility of fluorescent signals and that adequate washing steps have been taken to remove background staining. The most important considerations to achieve good staining results are the quality of the microscope, glass slides/cover slips, and the tissue sections. When viewing live purified neutrophils, the most important steps are in the purification process itself. Neutrophils are sensitive and can be activated by a number of different signals. In order to ensure that the experiment produces meaningful results, care must be taken to keep the purified neutrophils inactive until they are ready to be used. This can be achieved by performing the neutrophil purification protocol in an anoxic chamber to limit neutrophil exposure to oxygen.
Both a limitation and advantage of MUB40, depending upon the experimental question being addressed, is that MUB40 only stains activated neutrophils unless they are somehow permeabilized. This can be a huge advantage if the experiment calls for following inflammation in a live animal or purified cell, but it may be a disadvantage if the experiment requires detection of all live neutrophils regardless of activation state. A second potential limitation to this protocol is the fact that lactoferrin is present in various bodily secretions such as tears, milk, and mucus. For histopathology staining in which these secretions are maintained (e.g., colonic biopsies in which the mucus layer is intact), MUB40 will also stain the mucus layer along with any present neutrophils. In practice, this has yet to affect our analysis, but it should be noted as a potential signal source.
Currently, MUB40 is the only neutrophil biomarker that can differentiate between activated and non-activated live neutrophils. Also, MUB40, unlike antibody detection methods, does not appear to alter the function or survival of neutrophils in vitro or in vivo. For example, addition of specific antibodies against live neutrophils can be an activating signal affecting neutrophil function or survival in the host. This makes MUB40 a very useful reagent for research involving neutrophil activation or granule release in vitro or in vivo. Additionally, MUB40 has a broad host specificity range and can be directly conjugated to a number of different fluorophores, allowing for use in single-step staining assays across multiple hosts. Thus, MUB40 staining is comparable between mouse, human, and other mammalian targets, and it simplifies and reduces the number of reagents needed to detect neutrophils.
While this protocol focuses specifically on histopathology and live cell imaging using MUB40, we have also successfully used the peptide in FACS sorting and live animal in vivo imaging. We anticipate that MUB40 will be amenable to many different assays involving the detection of neutrophils or visualization of inflammatory events in the body, and that future studies and protocols will be developed to further leverage the spectrum of MUB40 uses, particularly using non-invasive imaging technics.

Neutrophils are one of the primary arms of the innate immune system and are routinely recruited to the sites of inflammation around the body. The study of neutrophils has been largely impaired by their short lifespan in vitro (less than 8 h) and by limited detection tools under basal conditions or after activation. Here, we present a well-tested protocol for the broad and specific detection of mammalian neutrophils in fixed/permeabilized samples. We also provide a detailed protocol for staining live neutrophils with MUB40. Using the live neutrophil staining protocol, the timing and location of neutrophil activation can be pinpointed. This protocol is ideal for researchers who wish to study neutrophil activation or granule release. Beyond their antimicrobial functions, neutrophils are now appreciated as immunomodulatory cells involved in a wide range of diseases and immune responses (innate and adaptive)1,2. Neutrophils are present in most inflammatory tissues, at high numbers in infected tissues, in inflammatory tumors3, during IBS and Crohn&#39;s flare-ups4, and in areas of non-infectious inflammation such as the synovia of Rheumatoid Arthritis (RA) patients5. Neutrophils contain four classes of pre-formed granules named azurophil (&#945;), specific (&#946;1), tertiary (&#946;2) granules, and secretory vesicles (&#947;)6. Upon migration to an inflammatory site, recruited neutrophils become activated and sequentially secrete granule contents (composed of adhesion, antimicrobial compounds and immunomodulatory molecules), which promotes further recruitment and contributes to infection resolution (but also to host tissue damage)7. While neutrophils are considered a critical aspect of innate immunity, to date there are few detection reagents available to study them, and even fewer that can be used to assess the activation state of living neutrophils.
Current methods of detecting neutrophils rely on monoclonal antibodies generated against cell-surface exposed antigens, such as Ly-6G2&#160;or proteins specifically stored in neutrophil granules under basal conditions (e.g., myeloperoxidase, lactoferrin). Advantages of monoclonal antibodies include their strong binding, sensitivity, and versatility under various assay conditions. However, there are several downsides to using monoclonal antibodies and anti-Ly-6G in particular. These downsides include the specificity, as Ly-6G is present on a majority of myeloid cells in bone marrow and on all granulocytes including eosinophils; thus, deciphering neutrophils from eosinophils with this marker requires more complexes approaches8. Another frequent tradeoff with monoclonal antibodies is their often-limited host specificity range, making comparison studies with more than one animal species difficult. A third drawback of antibody detection methods, especially when using live cells or in vivo, is their potential to disrupt cell function or lead to cell activation. For example, the administration of anti-Ly-6G to mice is commonly applied to neutrophil depletion and transient neutropenia9. It has additionally been demonstrated that antibody injection may stimulate neutrophil antitumor function10. Finally, antibody detection of neutrophils does not reveal the activation state of the cell.
We have identified a 40-amino acid peptide called MUB40, which can be used in a number of assays to label neutrophils under in vitro or in vivo conditions as well as assay the activation status of live neutrophils11. MUB40&#160;is derived from MUB7012, a domain of the Lactobacillus reuteri surface mucus-binding protein originally described for its ability to bind mucus13,14. MUB40&#160;interacts with glycosylated lactoferrin that is present at high concentrations in neutrophil-specific and tertiary granules. MUB40 can be exposed to these granules through standard permeabilization steps present in histopathology or fluorescence-activated cell sorting (FACS) protocols for robust staining of fixed neutrophils. When live neutrophils are kept in an inactivated state, the MUB40&#160;peptide is excluded from the granule contents, and cells do not stain positive with the marker. Upon activation, MUB40&#160;can bind to exposed lactoferrin and lead to robust staining with the peptide. Thus, MUB40 can be used to determine the activation state of purified neutrophils, making it an attractive marker to follow the infectious process. The ability to bind to live activated neutrophils also allows MUB40&#160;to be used as a tool to detect areas of neutrophil inflammation in live animal models (e.g., a mouse arthritis model15). The protocols in this manuscript detail how fluorescently labeled MUB40 can be used to reveal neutrophils in histology tissues and how to assay the activation of live purified neutrophils in vitro.

<table border="0" cellpadding="0" cellspacing="0" style="width:895px;" width="894">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">MUB40-Cy5</td>
			<td style="width:123px;">Benoit Marteyn</td>
			<td style="width:344px;">benoit.marteyn@pasteur.fr</td>
			<td style="width:167px;">Fluorescent MUB40 peptide (available with other conjugated fluorophores)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RI-MUB40-Cy5</td>
			<td>Benoit Marteyn</td>
			<td>benoit.marteyn@pasteur.fr</td>
			<td>Retro-inverso fluorescent MUB40 peptide. Synthesized with D-amino acids and resistant to proteases (available with other conjugated fluorophores)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Parformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td align="right">16005</td>
			<td>Fixative for histology</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S7903</td>
			<td>Solution used to remove excess PFA</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Optimal Cutting Temperature Compound (OCT)</td>
			<td>Sakura Finetek USA</td>
			<td align="right">4583</td>
			<td>Used to freeze tissue before cryostat sectioning</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">2-Methylbutane</td>
			<td>Sigma-Aldrich</td>
			<td>M32631</td>
			<td>Used to freeze tissue before cryostat sectioning</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td align="right">1.00974</td>
			<td>Used for dry ice bath to freeze tissue</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Leica Cryostat</td>
			<td>Leica Biosystems</td>
			<td>CM1520</td>
			<td>Used to section tissues</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass microscopy slides</td>
			<td>Fisher Scientific</td>
			<td>12-518-101</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cover slips</td>
			<td>Thor Labs</td>
			<td>CG15CH</td>
			<td>Hi precision coverslip</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dako pen&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>Z377821</td>
			<td>Used to prevent liquid loss during tissue staining</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Saponin</td>
			<td>Sigma-Aldrich</td>
			<td align="right">47036</td>
			<td>Used to permeabilize cells for IF staining</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DAPI</td>
			<td>Sigma-Aldrich</td>
			<td align="right">10236276001</td>
			<td>Stains DNA&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Alexa fluor 488 Phalloidin&#160;</td>
			<td>Fisher Scientific</td>
			<td>A12379</td>
			<td>Binds actin</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Prolong Gold antifade Mountant</td>
			<td>Fisher Scientific</td>
			<td>P36930</td>
			<td>Prolongs IF signal and resistance to photobleaching</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fluorescent microscope</td>
			<td>Various</td>
			<td>Various</td>
			<td>Used to image IF slides</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anoxic Cabinet</td>
			<td>Various</td>
			<td>Various</td>
			<td>Used for the purification of live inactivated neutrophils</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium Chloride NaCL</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E9884</td>
			<td>Washing buffer component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MACS BSA buffer</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td>Washing buffer component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Percoll</td>
			<td>Sigma-Aldrich</td>
			<td>P4937</td>
			<td>Gradient for neutrophil purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD235a glycophorin magnetic microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-050-501</td>
			<td>Used to remove contaminating RBCs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>Used in the removal of RBCs from neutrophils</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI-1640 without Phenol Red</td>
			<td>Sigma-Aldrich</td>
			<td>R8755</td>
			<td>Used for neutrophil assays</td>
		</tr>
	</tbody>
</table>

the mub40 peptide for use in detecting neutrophil-mediated inflammation events

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high concentrations in specific and tertiary granules of neutrophils. This protocol details how MUB40&#160;conjugated directly to a fluorophore can be used to stain neutrophils in fixed/permeabilized tissues as well as how this can be used in live-cell imaging to assay for neutrophil activation and de-granulation. Neutrophil detection methods are limited to species-specific monoclonal antibodies, which are not always suitable for certain applications. MUB40 does not penetrate the cell membrane and is thus excluded from lactoferrin stored in non-activated/non-permeabilized neutrophils. MUB40 has the added benefit of recognizing lactoferrin from a broad host range, making it especially useful for comparing results in studies involving multiple research models, reducing the number of duplicate reagents, and simplifying protocols through single-step staining.

Results of MUB40-stained tissues from histopathology slides typically reveal individual cells scattered throughout the tissue. MUB40&#160;stains lactoferrin, which is present in neutrophil granules and compartmentalized. Thus, typically seen are punctate staining or several large separated areas of signal coming from individual neutrophils (Figure 1). It is helpful to add a second cell marker such as DAPI to help co-localize the MUB40&#160;signal with the stained cell. The total number of detected cells depends upon the number of neutrophils present in the field of view and can vary dramatically depending upon the source and strength of the immune response. Shown here are representative images from purified fixed human neutrophils (Figure 1A) and from a tissue biopsy of an ulcerative colitis patient (Figure 1B). Also shown are images taken from a relatively low level of neutrophils in the lungs of mice infected with Klebsiella pneumoniae (Figure 1C) and a high level of neutrophils in the colon of a guinea pig infected with Shigella sonnei (Figure 1D).
Results using live, purified neutrophils typically show little-to-no cell staining at early time points, and gradually increase in RI-MUB40&#160;staining over time as more neutrophils become activated. Shown here is a time course series of images from an experiment using purified human neutrophils infected with fluorescent S. sonnei (Figure 2). Depending on the strength of the activating signal, neutrophils may begin staining with MUB40&#160;rapidly, so it is recommended to start acquiring images prior to the addition of activators. When cells become activated and RI-MUB40 positive, they should exhibit a similar staining profile to that of fixed and permeabilized cells, which give a punctate staining. The optimal concentration of MUB40 for both live and fixed cell staining is 1 &#956;g/mL (Figure 3). Lower concentrations (0.1-0.01 &#956;g/mL) can be used but result in lower signal intensity. It is also recommended to use a DIC image or other live-cell stain to differentiate which cells are displaying an RI-MUB40 signal.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58367/58367fig1.jpg" /> 
Figure 1: MUB40-stained neutrophils of human, mouse, and guinea pig origin. (A) Representative MUB40&#160;staining (green) of neutrophils purified from healthy human donors. Cell nuclei are stained with DAPI (blue). (B) Staining of human neutrophils in tissue from an ulcerative colitis biopsy. Neutrophils are revealed with MUB40&#160;(green) and cell nuclei are stained with DAPI (blue). (C) Histopathology from the lung of a mouse infected with Klebsiella pneumoniae. Mouse neutrophils are revealed in the tissue using MUB40&#160;(green) and cell nuclei are stained with DAPI (blue). (D) Histopathology from the colon of a guinea pig infected with Shigella sonnei. Neutrophils responding to the infection are revealed in the tissue with MUB40&#160;(green). S. sonnei bacteria (red) express the fluorescent dsRED protein. Cell nuclei are stained with DAPI (blue). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58367/58367fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58367/58367fig2.jpg" /> 
Figure 2: Live neutrophils stain with MUB40 only when activated. Selected images from a time lapse series involving purified human neutrophils infected with S. sonnei in the presence of RI-MUB40. In the first set of images (top) a neutrophil encounters a S. sonnei bacterium (red) shown with a green arrow. Over the time course, the bacterium is internalized by the neutrophil and digested. As the neutrophil becomes activated from the bacteria, it stains progressively stronger with RI-MUB40. Images on the left are fluorescent channels overlaid with DIC images. Images on the right are fluorescent channels only. Images were taken at 5-min intervals. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58367/58367fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58367/58367fig3.jpg" /> 
Figure 3: Effect of different MUB40 concentrations on neutrophil staining. Representative staining of fixed/permeabilized neutrophils with various concentrations of MUB40-Cy5. Purified human neutrophils were fixed and stained with the indicated concentrations of MUB40-Cy5. Cell nuclei are stained with DAPI (blue). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58367/58367fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events at JoVE.com

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

Here, we present a protocol to detect the presence of neutrophils in fixed/permeabilized histology sections and assess the activation state of live purified neutrophils. In particular, the MUB40&#160;peptide binds lactoferrin present in neutrophil-specific and tertiary granules. Exposure of the granule contents through either permeabilization or neutrophil activation allows for the marking of neutrophils.

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high ...

the-mub40-peptide-for-use-detecting-neutrophil-mediated-inflammation

Nanyang Technological University

Research

JoVE Journal

Biology

6.0K Views.  Institut Pasteur. Here, we present a protocol to detect the presence of neutrophils in fixed/permeabilized histology sections and assess the activation state of live purified neutrophils. In particular, the MUB40 peptide binds lactoferrin present in neutrophil-specific and tertiary granules. Exposure of the granule contents through either permeabilization or neutrophil activation allows for the marking of neutrophils.

Video: The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

The use of Biofeedback in Clinical Virtual Reality: The INTREPID Project

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life activities. Its high prevalence in general population and the severe limitations it causes, point out the necessity to find new efficient strategies to treat it. Together with the cognitive-behavioral treatments, relaxation represents a useful approach for the treatment of GAD, but it has the limitation that it is hard to be learned. The INTREPID project is aimed to implement a new instrument to treat anxiety-related disorders and to test its clinical efficacy in reducing anxiety-related symptoms. The innovation of this approach is the combination of virtual reality and biofeedback, so that the first one is directly modified by the output of the second one. In this way, the patient is made aware of his or her reactions through the modification of some features of the VR environment in real time. Using mental exercises the patient learns to control these physiological parameters and using the feedback provided by the virtual environment is able to gauge his or her success. The supplemental use of portable devices, such as PDA or smart-phones, allows the patient to perform at home, individually and autonomously, the same exercises experienced in therapist's office. The goal is to anchor the learned protocol in a real life context, so enhancing the patients' ability to deal with their symptoms. The expected result is a better and faster learning of relaxation techniques, and thus an increased effectiveness of the treatment if compared with traditional clinical protocols.

The Project on Virtual Reality Intelligent Multi-sensor System for the Treatment of Anxiety-related Disorders (INTREPID) is aimed at developing a multi-sensor context-aware virtual reality system for the treatment of anxiety-related disorders.

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine disruption. Estrogenic ligands include both natural and manmade &#34;Environmental Estrogens&#34; (EEs) found in many consumer goods including Personal Care Products (PCPs), plastics, pesticides, and foods. EEs disrupt hormone signaling in humans and other animals, potentially reducing fertility and increasing disease risk. Despite the importance of EEs and other Endocrine Disrupting Chemicals (EDCs) to public health, endocrine disruption is not typically included in undergraduate curricula. This shortcoming is partly due to a lack of relevant laboratory activities that illustrate the principles involved while also being accessible to undergraduate students. This article presents an optimized YES for quantifying ligands in personal care products that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates one of the two colorimetric substrates (ortho-nitrophenyl-&#946;-D-galactopyranoside (ONPG) or chlorophenol red-&#946;-D-galactopyranoside (CPRG)) that are cleaved by &#946;-galactosidase, a 6-day refrigerated incubation step to facilitate use in undergraduate laboratory courses, an automated application for LacZ calculations, and R code for the associated 4-parameter logistic regression analysis. The protocol has been designed to allow undergraduate students to develop and conduct experiments in which they screen products of their choosing for estrogen mimics. In the process, they learn about endocrine disruption, cell culture, receptor binding, enzyme activity, genetic engineering, statistics, and experimental design. Simultaneously, they also practice fundamental and broadly applicable laboratory skills, such as: calculating concentrations; making solutions; demonstrating sterile technique; serially diluting standards; constructing and interpolating standard curves; identifying variables and controls; collecting, organizing, and analyzing data; constructing and interpreting graphs; and using common laboratory equipment such as micropipettors and spectrophotometers. Thus, implementing this assay encourages students to engage in inquiry-based learning while exploring emerging issues in environmental science and health.

This article presents an optimized yeast estrogen screen for quantifying ligands in Personal Care Products (PCPs) that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates two colorimetric substrate options, a six-day refrigerated incubation for use in undergraduate courses, and statistical tools for data analysis.

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine ...

Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR

Lyme disease is a serious vector-borne infection that is caused by the Borrelia burgdorferi sensu lato family of spirochetes, which are transmitted to humans through the bite of infected Ixodes ticks. The primary etiological agent in North America is Borrelia burgdorferi sensu stricto. As geographic risk regions expand, it is prudent to support robust surveillance programs that can measure tick infection rates, and communicate findings to clinicians, veterinarians, and the general public. The molecular technique of nested polymerase chain reaction (nPCR) has long been used for this purpose, and it remains a central, inexpensive, and robust approach in the detection of Borrelia in both ticks and wildlife. 
This article demonstrates the application of nPCR to tick DNA extracts to identify infected specimens. Two independent B. burgdorferi targets, genes encoding Flagellin B (FlaB) and Outer surface protein A (OspA), have been used extensively with this technique. The protocol involves tick collection, DNA extraction, and then an initial round of PCR to detect each of the two Borrelia-specific loci. Subsequent polymerase chain reaction (PCR) uses the product of the first reaction as a new template to generate smaller, internal amplification fragments. The nested approach improves upon both the specificity and sensitivity of conventional PCR. A tick is considered positive for the pathogen when inner amplicons from both Borrelia genes can be detected by agarose gel electrophoresis.

Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR

Nested PCR is a sensitive, specific, and straightforward technique that can be applied to tick DNA extracts to probe for Borrelia burgdorferi, the causative agent of Lyme disease. The initial PCR experiment uses gene-specific primers to generate long amplicons, which then become templates for a subsequent reaction using internal primers.

Lyme disease is a serious vector-borne infection that is caused by the Borrelia burgdorferi sensu lato family of spirochetes, which are transmitted to ...