Funding from&#160;P30 DK034854&#160;supported VY, LB and studies in the Massachusetts Host-Microbiome Center and funding from NIH/NEI&#160;R01 EY022054 supported MG.

All procedures involving mice follow the Institutional Animal Care and Use Committee guidelines. Follow laboratory safety guidelines (as directed by your Institutional Environmental Health and Safety department) when working with microorganisms and potentially contaminated materials. Use appropriate waste receptacles and decontamination procedures prior to disposal of potentially biohazard contaminated materials.
1. Eye swab preparation, work field set-up, mouse eye swabbing and sample enrichment
<ol>
	<li>Prepare sterile eye swabs 
	NOTE: Sterile eye swabs are thinly coated wooden toothpicks with a fiber thin layer of cotton batting and made in house.
	<ol>
		<li>Autoclave appropriate amount of cotton batting and toothpicks for number of mice to be swabbed.</li>
		<li>Pinch off a half a centimeter-long piece of cotton batting with thumb and forefinger. Tease out batting by pulling on the edges with thumbs and forefingers to form a flat single porous layer, stopping just before the batting falls apart (small bits of cotton dispersed throughout the stretched batting are acceptable).</li>
		<li>Swirl the batting around one of the sharp ends of the toothpick by lightly holding the stretched-out piece on the toothpick tip as it is twisted, see Figure 1. The &#8220;completed&#8221; eye swab will have a very thin layer of cotton stretched over the tip, extending approximately one half to one centimeter away from the tip.</li>
		<li>Insert &#8220;completed&#8221; swabs into small beaker, swab side down and cover. Autoclave.</li>
	</ol>
	</li>
	<li>Set up the workspace
	<ol>
		<li>Prepare anesthesia containing 2 mL of ketamine (100 mg/mL), 400 &#181;L of xylazine (100 mg/ml) and 27.6 mL of sterile saline (0.9% NaCl in dH2O) in a sterile 50 mL sterile centrifuge tube. Filter sterilize and aliquot anesthesia for immediate use (may be stored at 4 &#176;C for 1 month; always allow to equilibrate to room temperature prior to use).</li>
		<li>Clear and clean the work area with disinfectant to minimize contamination.</li>
		<li>Aliquot 0.5 mL of sterile Brain Heart Infusion media (BHI) into labeled 1.5 mL sterile microcentrifuge tubes (1 tube per mouse and control); arrange in microcentrifuge rack. Set the rack on ice.</li>
		<li>Set up work flow as follows (from left to right): mouse anesthetizing station (cage containing experimental mice, empty sterile cage, room temperature anesthesia, 25 G needle and 1 mL syringe), eye swabbing station (aliquoted BHI on ice, sterilized eye swabs,&#160;clean paper towels, 70% isopropanol spray) and plating station (room temperature blood agar plates, 10 &#181;L pipette, 10 &#181;L sterile disposable tips, biohazard waste container).</li>
	</ol>
	</li>
	<li>Eye swabbing
	<ol>
		<li>Administer 10 &#181;L of anesthesia per 1 g of mouse weight dosage of ketamine and xylazine respectively intraperitoneally to each adult mouse and place into an autoclaved cage. Test anesthetization by squeezing&#160;hindfoot pad; no movement indicates appropriate anesthetization.</li>
		<li>Assign one hand to only handle anesthetized mice and the other hand to only handle the eye swab and the culture. For swabbing, remove the fully anesthetized mouse from the cage; place on top of clean work surface&#160;positioned on its&#8217; side with left eye exposed. Spray gloved hands with isopropanol, dry with clean paper towel.</li>
		<li>Uncap specifically labeled BHI micro-centrifuge tube with dedicated media handling hand. Place the tube back into the micro-centrifuge rack on ice.&#160; Dip the cotton coated tip of the eye swab in BHI, withdraw the eye swab from the tube swirling the tip 2 times against the inner tube to remove excess liquid.&#160;</li>
		<li>With the mouse handling hand, gently hold the mouse by the scruff of the neck. With the other hand, place the tip of eye swab against the medial conjunctival region of the left eye, see Figure 2A. Lightly depress the eyeball and move the swab in a window washing motion (Figure 2B) back and forth, between the lower eyelid and eye, 10 times. Maintain constant pressure.</li>
		<li>Without touching the fur, gently remove the tip of swab, perpendicular to where it was inserted, and place the swab cotton side down directly into a labeled micro-centrifuge tube containing BHI media.&#160;Apply a lubricating eye drop to the swabbed eye.</li>
		<li>Return the mouse to the cage.</li>
		<li>Let the swab stand for 10 to 15 min on ice and remove eye swab with the sterilized gloved hand by mixing the tip in the media for 10 rotations. Withdraw the swab by swirling tip against the inner wall of the micro-centrifuge tube for 5 rotations. Dispose of the swab in biohazard container.</li>
		<li>Repeat steps 1.3.2 to 1.3.7 for each mouse and for control samples (skin or fur swab). Sterilize gloves appropriately between each swab.</li>
	</ol>
	</li>
	<li>Enrichment
	<ol>
		<li>Enrich the sample by incubating the tube statically for 1 h at 37 &#176;C</li>
		<li>During incubation, label one room temperature Trypticase Soy with 5% sheep&#8217;s blood agar plate (TSA plate) per mouse and divide in half.</li>
		<li>After eye swab culture incubation, remove enriched samples from the incubator and place on ice.</li>
		<li>Briefly vortex samples to mix and plate according to the schematic in Figure 3A. Aliquot 10 &#181;L of the sample onto the TSA plate and tilt the plate to form a strip, repeat 2 times.</li>
		<li>On the other side of the plate&#8217;s dividing line, dot 10 &#181;L of sample on agar, repeat 9 times.</li>
		<li>Incubate plates at 37 &#176;C for 18 h, 2 days, and 4 days (in a clean chamber that prevents agar plates from drying). Count colonies in strips, note morphology and calculate colony forming units (CFUs) per swab for morphologically similar isolates. Look at dots for unique organisms not captured in strips.</li>
	</ol>
	</li>
</ol>
2. Master plate, characterization, and identification of ocular microbes
<ol>
	<li>Master plate
	<ol>
		<li>Draw grids on the back of a TSA plate (Figure 3B). Pick a colony with a sterile toothpick from each mouse eye swab plate and streak the master plate square (grids), label grid with colony number and mouse information. Pick and plate three different colonies for each morphologically distinct colony.</li>
		<li>Incubate the plate overnight at 37 &#176;C. Continue incubation for 96 hours, checking daily for the appearance of new isolates. 
		NOTE: The Commensal population will vary based on mouse age, nutrition, disease state and caging practices. Isolate characterization is based on the predominant aerobic and facultative anaerobic bacteria found in our laboratory.</li>
	</ol>
	</li>
	<li>Characterization
	<ol>
		<li>Duplicate13 plate microbes on Mannitol Salt Agar (MSA) and MacConkey (MAC) plates, incubate overnight at 37 &#176;C. Note growth for each isolate and presence of waxy colonies or yellow colonies with halo on MSA.</li>
		<li>For Gram positive cocci, test with the catalase test13,14. Place a drop of 3% hydrogen peroxide on a clean glass slide. Using a sterile toothpick, pick the corresponding microbe from the eye swab plate. No bubble formation indicates Streptococcus spp., slight bubble formation indicates Corynebacterium spp. and perhaps Aerococcus spp. and rapid bubble formation indicates Staphylococcus spp.</li>
	</ol>
	</li>
	<li>Identification: MALDI-TOF MS analysis 
	NOTE: Bacterial identifications are carried out using the MALDI-TOF and the Software Database. This system employs matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for developing spectra profiles which are compared to known bacterial spectra for identification. E.coli, ATCC 8739, is used for system calibration.
	<ol>
		<li>Plate bacterial isolates onto TSA plates containing 5% sheep blood and incubate overnight with 5% carbon dioxide at 37 &#730;C.</li>
		<li>Using a 1 &#181;L loop, apply a thin layer of the pure bacteria to the MALDI-TOF target slide; 1 &#181;L of MALDI-TOF MS CHCA matrix solution (alpha-cyano-4-hydroxycinnamic acid) is overlaid and allowed to air dry completely before loading the slide into the MALDI-TOF instrument.</li>
		<li>Each isolate is spotted in duplicate.</li>
		<li>Reports containing probability scores greater than 80%, which indicate a high discrimination value, are accepted as reliable results and the bacterial identification accepted.</li>
	</ol>
	</li>
</ol>

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No conflict of interest to disclose.

Due to the paucibacterial state of the ocular surface, many laboratories have had difficulty isolating ocular commensals7,20, resulting in low number of samples with growth, low abundance and low diversity8. This method significantly improves upon standard culture practices4,21 by the addition of an enrichment step, as well as a redesigned eye swab and identification by MALDI-TOF M...

The aim of this protocol is to enhance specific isolation of viable and rare aerobic and facultative anaerobic microbes from the ocular conjunctiva to characterize the ocular microbiome. Extensive studies have profiled commensal mucosal communities on the skin, gut, respiratory and genital tracts and show that these communities influence the development of the immune system and response1,2,3. Ocular commensal communities have been shown to change during certain disease pathologies, such as Dry eye disease4, Sjogren&#8217;s syndrome5 and diabetes6. Yet, the ability to define a typical ocular surface commensal community is hampered by their relatively low abundance compared to the other mucosal sites6,7,8. This prompts a controversy over whether there is a resident ocular microbiome and if it exists, whether it differs from the skin microbiome and consequently, its local effect on the innate immune system development and response. This protocol can help resolve this question.
Generally, approaches to define the ocular commensal niche are based on sequencing and culture-based techniques4,7,9. 16 S rDNA sequencing and BRISK analysis7 show a broader diversity than culture-based techniques, but are unable to differentiate between live and dead microbes. Since the ocular surface is hostile to many microbes due to tear film&#8217;s anti-microbial properties4 generating a large array of DNA fragments, DNA based approaches will detect these artifacts which may skew the data toward identification of dead bacteria as resident commensals rather than contaminants. This results in aberrant commensal identification and characterization of the ocular space as being higher in microbe abundance and diversity10. This makes it difficult to define the resident ocular microbiome via DNA based methods. Whereas, standard culture-based techniques are unable to detect commensals because the load is too low11. Our method improves upon standard practices by using a thin swab that can target the conjunctiva, thus avoiding contamination from neighboring skin, as well as the concept that viable organisms can be enriched by brief culture in nutrient dense media with the goal of resuscitating viable but non-culturable, as well as, enriching for rare viable microbes.
The results, relative abundance of ocular commensals per eye swab, characterize the conjunctiva resident microbiome and are important for comparative purposes. Our data shows that there is a difference between skin and conjunctival microbiota, as well as greater diversity with increased age and a sex specific difference in abundance. Furthermore, this approach has reproducibly found commensal differences in knock-out mice12. This protocol can be applied to describe the ocular microbiome which may vary due to caging practices, geography, or disease state, as well as the local effects of commensal metabolites and products on immune system development and response.

<table>
	<tbody>
		<tr>
			<td>0.1 to 10 &#181;l pipet tip</td>
			<td>USA Scientific</td>
			<td>1110-300</td>
			<td>autoclave before use</td>
		</tr>
		<tr>
			<td>0.5 to 10 &#181;l Eppendorf pipet</td>
			<td>Fisher Scientific</td>
			<td>13-690-026</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>Fisher Scientific</td>
			<td>BD309623</td>
			<td>1 syringe for each eye swab group</td>
		</tr>
		<tr>
			<td>1.5 ml Eppendorf tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td>autoclave before use</td>
		</tr>
		<tr>
			<td>1000 &#181; ml pipet tip</td>
			<td>USA Scientific</td>
			<td>1111-2021</td>
			<td>autoclave before use</td>
		</tr>
		<tr>
			<td>200 to 1000&#181;l Gilson pipetman (P1000)</td>
			<td>Fisher Scientific</td>
			<td>F123602G</td>
			<td></td>
		</tr>
		<tr>
			<td>25 G needle</td>
			<td>Fisher Scientific</td>
			<td>14-826AA</td>
			<td>1 needle per eye swab group</td>
		</tr>
		<tr>
			<td>3 % Hydrogen Peroxide</td>
			<td>Fisher Scientific</td>
			<td>S25359</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176; C Incubator</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70 % Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>PX1840-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ana-Sed Injection (Xylazine 100 mg/ml)</td>
			<td>Santa Cruz Animal Health</td>
			<td>SC-362949Rx</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BBL Gram Stain kit</td>
			<td>Fisher Scientific</td>
			<td>B12539</td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen Burner</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clean paper towels</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Batting/Sterile rolled cotton</td>
			<td>CVS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable 1 ml Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-711-9AM</td>
			<td>for Gram stain and catalase tests</td>
		</tr>
		<tr>
			<td>E.coli</td>
			<td>ATTC</td>
			<td>ATCC 8739</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-A3</td>
			<td>for Gram stain and catalase tests</td>
		</tr>
		<tr>
			<td>Ketamine (100mg/ml)</td>
			<td>Henry Schein</td>
			<td>9950001</td>
			<td></td>
		</tr>
		<tr>
			<td>Mac Conkey Agar Plates</td>
			<td>Fisher Scientific</td>
			<td>4321270</td>
			<td>store at 4 &#176;C until ready to use</td>
		</tr>
		<tr>
			<td>Mannitol Salt Agar</td>
			<td>Carolina Biological Supply</td>
			<td>784641</td>
			<td>Prepare plates according to mfr&#39;s instructions, store at 4 &#176;C for 1 week</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Jackson Labs</td>
			<td>C57/BL6J</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>08-757-12</td>
			<td>for Mannitol Salt agar plates</td>
		</tr>
		<tr>
			<td>RPI Brain Heart Infusion Media</td>
			<td>Fisher Scientific</td>
			<td>50-488525</td>
			<td>prepare according to directions and autoclave</td>
		</tr>
		<tr>
			<td>SteriFlip (0.22 &#181;m pore size polyester sulfone)</td>
			<td>EMD/Millipore, Fisher Scientifc</td>
			<td>SCGP00525</td>
			<td>to sterilize anesthesia</td>
		</tr>
		<tr>
			<td>Sterile Corning Centrifuge Tube</td>
			<td>Fisher Scientific</td>
			<td>430829</td>
			<td>anesthesia preparation</td>
		</tr>
		<tr>
			<td>Sterile mouse cage</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tooth picks (round bamboo)</td>
			<td>Kitchen Essentials</td>
			<td></td>
			<td>autoclave before use and swab preparation</td>
		</tr>
		<tr>
			<td>Trypticase Soy Agar II with 5% Sheep&#39;s Blood Plates</td>
			<td>Fisher Scientific</td>
			<td>4321261</td>
			<td>store at 4 &#176;C until ready to use</td>
		</tr>
		<tr>
			<td>Vitek target slide</td>
			<td>BioMerieux Inc. Durham,NC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vitek-MS</td>
			<td>BioMerieux Inc. Durham,NC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vitek-MS CHCA matrix solution</td>
			<td>BioMerieux Inc. Durham, NC</td>
			<td>411071</td>
			<td></td>
		</tr>
		<tr>
			<td>Single use eye drops</td>
			<td>CVS Pharmacy</td>
			<td></td>
			<td>Bausch and Lomb Soothe Lubricant Eye Drops, 28 vials, 0.02 fl oz. each</td>
		</tr>
	</tbody>
</table>

conjunctival commensal isolation and identification in mice

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. Identification and monitoring of bacterial species at the ocular mucosa have been challenging due to their low abundance and limited availability of appropriate methodology for commensal growth and identification. There are two standard approaches: culture based or DNA sequencing methods. The first method is problematic due to the limited recoverable bacteria and the second approach identifies both live and dead bacteria leading to an aberrant representation of the ocular space. We developed a robust and sensitive method for bacterial isolation by building upon standard microbiological culturing techniques. This is a swab-based technique, utilizing an &#8220;in-lab&#8221; made thin swab that targets the lower conjunctiva, followed by an amplification step for aerobic and facultative anaerobic genera. This protocol has allowed us to isolate and identify conjunctival species such as Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp., etc. The approach is suitable to define commensal diversity in mice under different disease conditions.

Representative results for an eye swab plate demonstrating different methods for plating are pictured in Figure 3A showing morphologically diverse isolates from C57BL/6 mouse. For each distinct isolate, the colonies were counted in the strip and the relative abundance, unique Colony Forming Units (CFUs) per eye swab, calculated and plotted for comparison purposes. For microbiological characterization, bacteria were picked from individual mouse eye swab plates to produce a master TSA plate (c...

Watch this Scientific Journal Video about Conjunctival Commensal Isolation and Identification in Mice at JoVE.com

Conjunctival Commensal Isolation and Identification in Mice

Presented here is a protocol for the isolation and amplification of aerobic and facultative anaerobic mouse conjunctival commensal bacteria using a unique eye swab and culture-based enrichment step with subsequent identification by microbiological based methods and MALDI-TOF mass spectrometry.

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. ...

conjunctival-commensal-isolation-and-identification-in-mice

Research

JoVE Journal

Immunology and Infection

4.3K Views.  Harvard Medical School. Presented here is a protocol for the isolation and amplification of aerobic and facultative anaerobic mouse conjunctival commensal bacteria using a unique eye swab and culture-based enrichment step with subsequent identification by microbiological based methods and MALDI-TOF mass spectrometry.

Video: Conjunctival Commensal Isolation and Identification in Mice

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Isolation and Identification of Extravascular Immune Cells of the Heart

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

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

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.