This research was supported by the U.S. Department of Agriculture, Agricultural Research Service (USDA-ARS), National Program 108, Current Research Information System numbers 8072-42000-093 and 8072-42000-094-000-D. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U. S. Department of Agriculture. USDA is an equal opportunity provider and employer.

All work associated with this protocol should be conducted within a biological safety cabinet (BSC) to maintain aseptic conditions and minimize the risk of sample contamination or operator exposure to microbial pathogens. When transferring samples outside the BSC, use sealed containers to prevent spillage in case of accidental drops, maintaining sample integrity. Preferably, disposable components should be used throughout the procedure to mitigate the possibility of cross-contamination. In cases where disposables are not feasible, ensure all equipment and materials are sterile prior to use. Proper waste management is crucial; all used disposable components should be discarded as biohazard waste. Autoclave materials before discarding to ensure proper sterilization and avoid containment of potentially hazardous materials. Adhering to these precautions not only safeguards sample integrity but also minimizes the risk of operator exposure to microbial pathogens. Figure 1 depicts the workflow of sample preparation, selective enrichment, filter-based isolation, and mqPCR differentiation of Campylobacter species. Supplemental File 1 depicts a more detailed workflow and images throughout the process.
1. Preparation of meat samples
<ol>
	<li>Acquiring meat samples
	<ol>
		<li>Acquire various fresh meat packages, including chicken thighs, wings, drumsticks, and livers from local retailers.</li>
		<li>Transfer all samples to storage at 4 &#176;C and process within 24 h after receipt. 
		NOTE: Storing the fresh samples at lower temperatures, such as below freezing, will affect the recovery.</li>
	</ol>
	</li>
	<li>Processing meat samples
	<ol>
		<li>Follow the ratio of components prescribed within the FSIS sampling guideline6,17.</li>
		<li>Cut 450 g chicken pieces from each package and place them in a stomacher bag (see Table of Materials). 
		NOTE: Stomacher bags are suggested because they have sufficient mechanical strength to ensure the downstream processes, and will not rupture or leak.</li>
		<li>Prepare buffered peptone water (BPW).
		<ol>
			<li>Dissolve 20 g of the powder (see Table of Materials) in 1 L of purified water. Autoclave the solution at 121 &#176;C for 15 min. Dilute the solution in sterile water to a concentration of 0.1%.</li>
		</ol>
		</li>
		<li>Add 200 mL 0.1% BPW to the stomacher bag containing the chicken.</li>
		<li>Manually massage/palpate the sample from the outside of the stomacher bag for 2 min.</li>
		<li>Collect all the chicken rinse from the filtered side of the bag using a motorized pipette controller (see Table of Materials).</li>
		<li>Dispense the chicken rinse into sterile centrifuge bottles. Centrifuge at 10,000 x g for 10 min at room temperature.</li>
		<li>Carefully collect the supernatant using a motorized pipette controller with a 25 mL disposable serological plastic pipette. Avoid disturbing the pellet.</li>
		<li>Repeat the process as necessary to ensure all of the supernatant is removed.</li>
		<li>Discard the collected supernatant.</li>
	</ol>
	</li>
</ol>
2.&#160;Selective enrichment of Campylobacter from raw meat
<ol>
	<li>Preparation of Bolton Broth with supplements
	<ol>
		<li>Dissolve 13.8 g of powder (see Table of Materials) in 500 mL of purified water. Sterilize the broth by autoclaving for 15 min at 121 &#176;C.</li>
		<li>Add 25 mL laked horse blood (see Table of Materials) to the sterilized 500 mL Bolton Broth. 
		NOTE: The addition of horse blood acts as an oxygen quenching agent to aid in the recovery of injured Campylobacter cells from the food matrix.</li>
		<li>Reconstitute 1 vial of antibiotic supplement (cefoperazone, cycloheximide, trimethoprim, and vancomycin, see Table of Materials) in 5 mL 50% ethanol.</li>
		<li>Add the reconstituted antibiotic supplement to the Bolton Broth.</li>
	</ol>
	</li>
	<li>Enrichment procedure
	<ol>
		<li>Resuspend the pellet in 50 mL Bolton Broth containing laked horse blood and antibiotics.</li>
		<li>Place samples (with loosened caps) inside a sealed container that maintains a gas mixture of 85% N2, 10% CO2, and 5% O2.
		<ol>
			<li>Ensure the cap is loose, but the container is tightly sealed to produce the microaerophilic and thermophilic growth requirements of Campylobacter. 
			NOTE: Gas packs that maintain an atmosphere of 85% N2, 10% CO2, and 5% O2 can be used when environmental chambers are not available.</li>
			<li>Incubate the samples at 42 &#176;C for 24 h.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Isolation and purification of C. jejuni and C. coli from raw chicken
<ol>
	<li>Preparation of Brucella agar plates
	<ol>
		<li>Dissolve 28 g of Brucella powder (see Table of Materials) in 1 L of purified water. Dissolve 15 g of agar in the Brucella solution.</li>
		<li>Sterilize the Brucella agar by autoclaving for 15 min at 121 &#176;C. Cool the medium-agar mix in a 55 &#176;C water bath.</li>
		<li>Pour 20 mL of Brucella agar into each 100 mm diameter Petri dish.</li>
	</ol>
	</li>
	<li>Filter method and colony cultivation
	<ol>
		<li>Evaluate the effect of moisture on Campylobacter passing through filters by drying Brucella agar plates with lids opened in a Biosafety cabinet for 0 h, 1 h, 2 h and 3 h.</li>
		<li>Prepare a no-filter control by directly spreading 80 &#181;L of the sample on the Brucella agar plate.</li>
	</ol>
	</li>
	<li>Place a cellulose acetate filter (0.45 &#181;m or 0.65 pore-size, see Table of Materials) at the center of a Brucella agar plate.</li>
	<li>Pipette 4 drops/filter and 20 &#181;L/drop of enriched sample onto the filter.
	<ol>
		<li>Place the drops near the center of the filter to ensure the liquid that reaches the plate goes through the filter, not around the filter.</li>
		<li>Place the drops in a manner that ensures they will not spread and aggregate.</li>
	</ol>
	</li>
	<li>Incubate drops at room temperature for 15 min and carefully remove the filters. 
	NOTE: This step permits sufficient time for the Campylobacter cells to traverse the membrane and reach the agar medium without excessive drying.</li>
	<li>Incubate plates at 42 &#176;C for approximately 24 h under the microaerobic conditions described earlier.</li>
	<li>Pick characteristic Campylobacter colonies with specific traits. 
	NOTE: Campylobacter colonies are typically round with smooth edges, glistening, and translucent yellowish or pinkish color6.</li>
	<li>Streak colonies onto Brucella agar plates for purification. Repeat this step until plates with a single uniform colony morphology are obtained.</li>
	<li>Prepare samples for long-term storage.
	<ol>
		<li>Prepare Bolton Broth as described earlier. Add one colony from the plate with uniform colony morphology.</li>
		<li>Grow overnight (24 h) under microaerophilic conditions described earlier. Add 900 &#181;L of the overnight culture to a 2 mL cryovial containing 100 &#181;L of DMSO.</li>
		<li>Rapidly cool in a dry ice-ethanol bath (approx. -72 &#176;C) for 10 min. Transfer to a -80 &#176;C freezer for long-term storage.</li>
	</ol>
	</li>
</ol>
4. Identification of C. jejuni and C. coli species
<ol>
	<li>Perform species-level identification of C. jejuni and C. coli using a multiplex qPCR (mqPCR) assay previously developed18,19.
	<ol>
		<li>Perform rapid cell lysis and genomic DNA extraction in a 96-well plate format. 
		NOTE: It is strongly recommended to consider using commercial kits (see Table of Materials) to ensure that the sample is sufficiently free of known inhibitors of PCR.
		<ol>
			<li>Disperse purified Campylobacter colonies into 100 &#181;L of extraction solution.</li>
			<li>Lyse samples at 99 &#176;C for 10 min followed by cooling at 20 &#176;C for 2 min in a thermocycler.</li>
			<li>Centrifuge the plate at 8,000 x g for 10 min at room temperature. Remove 2 &#181;L of aliquots of the supernatant for the mqPCR assay.</li>
			<li>Prepare a 20 &#181;L reaction mixture consisting of 10 &#181;L of 2x Master Mix, 2.0 &#181;L of DNA sample, 104 copies of Internal Amplification Control (IAC) template, and 200 nM of each primer and probe (see Table of Materials). 
			NOTE: The primers and probes of hipO and cdtA are the exclusive target genes for C. jejuni and C. coli, respectively.&#160;The IAC consists of a 79-bp DNA segment of the human adenovirus and is included as a positive control to ensure consistent activity of DNA polymerase across all samples.</li>
		</ol>
		</li>
		<li>Load all samples in triplicates in a 96-well optical plate covered with an optical film and place them in a Real-Time PCR system (see Table of Materials).
		<ol>
			<li>Initiate a hot-start activation of the DNA polymerase at 95 &#176;C for 10 min.&#160;Follow with 40 cycles of denaturation at 95 &#176;C for 15 s and annealing/extension at 60 &#176;C for 1 min.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Enumerate cell suspensions
<ol>
	<li>Enumerate cell suspensions using a 6 x 6 drop plate procedure20. Refer to Supplemental File 1 for images depicting the 6 x 6 drop plate method.</li>
	<li>Air dry Brucella agar plates with the lid off in a laminar flow hood for 45 min.</li>
	<li>Add 200 &#181;L of bacterial suspension to six rows (A-F) in the first column of a 96-well plate.</li>
	<li>Distribute 180 &#181;L of Brucella medium across six rows of the remaining columns (2-12).</li>
	<li>Prepare ten-fold serial dilutions using 20 &#181;L transfers.
	<ol>
		<li>For instance, transfer 20 &#181;L of sample from column one to column two.&#160;Repeat this process for a minimum of 6 columns.</li>
		<li>Reflux mix each suspension ten times using the pipette, changing the tips between each transfer.</li>
	</ol>
	</li>
	<li>Use a multichannel pipette to deposit 7 &#181;L drops from six rows of a column on the surface of a Brucella agar plate.</li>
	<li>Repeat to create a 6 x 6 array, ensuring rows are technical replicates across columns.</li>
	<li>Air dry the plates for 5 min, and then invert the plate.Incubate plates at 42 &#176;C for 24 h.</li>
	<li>Count the number of colonies in each representative dilution.</li>
</ol>

Enhanced Campylobacter Isolation from Meat

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+dormancy+in+Campylobacter:+Abstract+theory+or+cause+for+concern.">Bacterial dormancy in Campylobacter: Abstract theory or cause for concern.</a> Int J Food Sci Technol. 36 (6), 593-600 (2001).</li><li>Saha, S., Saha, S., Sanyal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+injured+Campylobacter+jejuni+cells+after+animal+passage.">Recovery of injured Campylobacter jejuni cells after animal passage.</a> Appl Environ Microbiol. 57 (11), 3388-3389 (1991).</li><li>Baylis, C. L., Macphee, S., Martin, K. W., Humphrey, T. J., Betts, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+three+enrichment+media+for+the+isolation+of+Campylobacter+spp.+From+foods.">Comparison of three enrichment media for the isolation of Campylobacter spp. From foods.</a> J Appl Microbiol. 89 (5), 884-891 (2000).</li><li>Line, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+selective+differential+agar+for+isolation+and+enumeration+of+Campylobacter+spp.">Development of a selective differential agar for isolation and enumeration of Campylobacter spp.</a> J Food Prot. 64 (11), 1711-1715 (2001).</li><li>Armstrong, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impacts+of+clarification+techniques+on+sample+constituents+and+pathogen+retention.">Impacts of clarification techniques on sample constituents and pathogen retention.</a> Foods. 8 (12), 636 (2019).</li></ol>

All the authors declare that there is no conflict of interest.

Significance of the protocol 
C. jejuni and C. coli were the two major species of Campylobacter found to be prevalent in poultry22 and animal livers23,24. In this study, the meat samples of chicken parts (legs, wings, and thighs), chicken livers, and beef livers were randomly collected during different time periods, and from different retail stores and manufacturers for the isolation of Campylobacter spp. Of the 49 total Campylobacter strains isolated, 36 were identified as C. jejuni and 13 were C. coli, with no other Campylobacter species found, which is consistent with other reports25.
The assay is based on the spiral-shaped cell morphology and characteristic corkscrew-like motility of Campylobacter spp. A simple, yet effective, passive filtration technique26,27 that exploited its spiral-shaped cell morphology (long, slender, 0.2-0.9 by 0.5-5 &#181;m) and strong corkscrew motility was used to separate Campylobacter from a mixture of background organisms. The high motility of Campylobacter allowed the cells to traverse the membrane filters and move towards favorable conditions found within the agar medium, while other background microorganisms from the meat products were unable to pass through. This method is relatively inexpensive, rapid, and selective, which makes it suitable for use in a variety of settings, including food processing facilities, clinical laboratories, and research laboratories.
A pioneering article often cited states that the 0.45 &#181;m filter worked so well that 0.65 &#181;m was not evaluated28. Results from this present study indicate the 0.65 &#181;m pore size filter performed significantly better than the 0.45 &#181;m pore size, resulting in a 29-fold increase in the number of cells recovered from the enrichment. This is important because the filters selected do not display reduced selectivity as previously reported29. Further, as it is known that filtering will significantly reduce the amount of Campylobacter recovered compared to direct plating30, therefore, increasing the size of the pore improves recovery of the microorganism, which is consistent with previously reported findings21. This is significant because all the cells that traversed the filters formed uniform Campylobacter colonies, indicating that both filters were sufficient at preventing other microflora and food particles from passing through. Additionally, the FSIS flowchart7 notes the potential for extended result production due to re-streaking isolates on Campy-Cefex plates containing antibiotics. Contrastingly, the protocol described in this manuscript, which combines the use of filtration and selective enrichment with cefoperazone, cycloheximide, trimethoprim, and vancomycin, has not necessitated re-streaking.
The current method employed is consistent with current FSIS Sampling and Verification programs17. As the level of Campylobacter contamination can be low (153 CFU/450 g chicken), the rinse is centrifuged to concentrate the sample by a factor of four, which increases the sensitivity of the assay. After concentrating the rinsate by a factor of 4x, samples are enriched for 48 h and screened with the Molecular Detection System (MDS) to replicate the method employed by FSIS laboratories (data not shown). Notably, the method described has yet to fail to identify positive strains within 24 h that were detected by the Molecular Detection System using 48 h of enrichment (data not shown). Lastly, an additional benefit of this protocol is that it can provide information related to the bacterial species and identify if the Campylobacter is C.coli, C. jejuni, or C. lari, while the MDS adopted in MLG 41.07 can only provide a binary positive/negative response for Campylobacter.
Critical steps 
The protocol for Campylobacter isolation and identification necessitates precision during centrifugation, filtration, and molecular analysis. Accurate dilutions, proper incubation conditions, and meticulous adherence to qPCR assay conditions are pivotal for reliable species identification.
As a microaerophilic bacterium, Campylobacter is very fragile and sensitive to various environmental stresses and requires unique fastidious conditions for growth31,32,33. In food samples typically undergoing lengthy periods of transportation and storage, many Campylobacter cells are perhaps in a dormancy or sublethal/lethal injured state34,35. Thus, it is important to recover the stressed cells from their food matrices and grow them to a higher concentration. In the first step of the procedure, we used Bolton Broth supplemented with laked horse blood and antibiotics for selective enrichment of Campylobacter from food. The add-in blood served as an oxygen quenching agent to overcome the adverse effects of free oxygen radicals36. The antibiotics were used to inhibit the growth of background microflora37.
To minimize the exposure time of Campylobacter to ambient atmospheric oxygen, a 15 min incubation period was selected to allow for the cells to traverse the filter. Also, the moisture of the Brucella agar plate under the filter played an important role in the rate of passage. Specifically, the results from testing agar plates dried for 0 h, 1 h, 2 h and 3 h suggested that a high moisture content in the filter prevented cells from passing through. Equally critical is the precise placement of filters and drops on the plates and filters, both influencing the success of isolating cells.
Potential pitfalls and limitations 
While presenting a structured approach for isolating and identifying Campylobacter species from raw chicken samples, several limitations of this protocol deserve attention. External contamination, insufficiently dried plates, clogging of filters impeding microbial movement, entrapment of the microorganisms within the pellet, incomplete sealing of the atmospheric chamber, and drops spreading beyond filter boundaries are among the primary pitfalls.
Inadequate separation of the microorganisms from the food surfaces or their confinement within the bulk of the sample may hinder their isolation using this method. Additionally, relying on microbial motility for traversal through passive filters presents a notable limitation; it is possible that the filter membranes retained some less motile Campylobacter strains, as it has been shown filters can reduce the capture efficiency of microbial pathogens in food38. Further limitations encompass the batch nature of centrifugation and filtration processes, susceptibility to filter clogging, and inefficiency in dispersing the pellet formed, which will impact the accuracy of microbial loads. These limitations collectively emphasize the need for caution and supplementary methodologies in ensuring comprehensive analysis, especially when dealing with varied sample types or seeking high-throughput capabilities.
Suggestions for troubleshooting 
To preempt potential issues, initially ensure that all materials adhere to the necessary quality standards and have not expired. Troubleshoot clogged filters by potentially employing an additional filtration to remove any large contaminates that may restrict the passage of the Campylobacter through the nitrocellulose membrane. If contamination is observed, verify that the drops were not placed too close to the edge of the filter and permitted liquid to reach the agar by going around the filter as opposed to through the pores. If there is insufficient growth following enrichment, verify the seals of the atmospheric containers are tight and not leaking.
Potential refinement and expansion 
Exploring alternative filter materials may enhance microbial traversal and enable this protocol to be expanded for use in isolating other motile microorganisms from heterogeneous mixtures such as food. Identifying controls to retain less motile Campylobacter variants without negatively impacting the specificity is advisable. Additionally, while the multiplexed qPCR assay utilized in this study was demonstrated to have the capabilities to detect C.lari18 other Campylobacter species of interest can be included within this assay.
In summary, through evaluating different parameters and settings, the appropriate conditions for filter-based isolation and species-level identification of C. jejuni and C. coli from food were established. The method has been demonstrated to be sensitive, specific, robust, and cost-effective. By applying it to real food samples, the protocol was able to isolate 36 C. jejuni and 13 C. coli strains from 79 meat packages.
The protocol is aligned with FSIS Directive 10,250.117, which outlines the procedure for raw chicken part sampling, and MLG 41.076 for isolation and identification of Campylobacter. The data suggests that concentrating the sample by 4x and enriching it for 24 h, coupled with filtration and plating, yields isolated, confirmed colonies within 48 h as opposed to 96 h. The protocol is compatible with DNA-based methods such as genome sequencing to provide a comprehensive characterization of Campylobacter strains, including their antimicrobial resistance profiles, virulence predictions, and phylogenetic relationships. The protocol represents a promising alternative for the efficient recovery and isolation of Campylobacter spp. from raw poultry, which can facilitate epidemiological studies and public health interventions.

Campylobacter spp. are the leading cause of bacterial foodborne gastroenteritis worldwide, with an estimated 800 million cases annually1. As a major zoonotic bacterium, Campylobacter naturally colonizes the gastrointestinal tracts of a wide range of animals, including wild birds, farm animals, and pets2. During slaughtering or food processing, Campylobacter spp. frequently contaminate carcasses or meat products3. Campylobacteriosis is usually associated with the consumption of undercooked poultry or cross-contamination of other foods by raw poultry juices2. It can cause serious complications, such as Guillain-Barr&#233; syndrome, reactive arthritis, and septicemia in immunocompromised individuals4. Detecting and isolating Campylobacter from food sources, especially poultry products, is essential for public health surveillance, outbreak investigation, and risk assessment.
Conventional culture-based methods are the traditional and standard methods for Campylobacter detection5,6. However, there are several limitations, including long incubation times (48 h or more), low sensitivity (up to 50%), and are not inclusive to all strains (some stressed Campylobacter cells may not grow well or at all in the media)7. Molecular methods, such as polymerase chain reaction (PCR), are more rapid and sensitive than culture-based methods, but they do not provide viable isolates for further characterization8,9.
Immunological methods are alternative and complementary methods for Campylobacter detection. These are rapid, simple, and versatile, but also have several limitations, including cross-reactivity (some antibodies may bind to non-Campylobacter bacteria or other substances that share similar antigens), low specificity (some antibodies may not bind to all Campylobacter strains or serotypes), and sample preparation requirements (immunological methods often require pre-treatment of the samples to remove interfering substances to enhance the binding of the antibodies)10.
Within the genus of Campylobacter, C. jejuni and C. coli cause most human Campylobacter infections (81% and 8.4%, respectively)11. Both are spiral-shaped, microaerophilic, and thermophilic bacteria containing a unipolar flagellum or bipolar flagella. Rotation of a flagellum at each pole is considered both the primary driving force for its characteristic corkscrew motility and crucial to its pathogenesis because it allows the bacterium to swim through the viscous mucosa of the host gastrointestinal tract. The motility of Campylobacter is controlled by its chemosensory system that allows the cells to move toward favorable environments12,13. Based on the cell morphology and physiological characteristics of Campylobacter, a few studies have utilized membrane filtration for the isolation of Campylobacter spp. from fecal and environmental samples14,15,16.
This study presents a rapid and robust protocol for the isolation and subsequent detection of C. jejuni and C. coli from raw meat, which overcomes the drawbacks of the existing methods and offers several advantages. Tentative colonies can be confirmed as Campylobacter spp. using a variety of methods, such as microscopy, biochemical tests (e.g., catalase and oxidase activity assays), or molecular methods6. The method identifies the isolates at the species level using a multiplex real-time PCR (mqPCR) assay that targets genes unique to C. jejuni and C. coli. This method is relatively inexpensive, rapid, and selective, which makes it suitable for use in a variety of settings, including food processing facilities, clinical laboratories, and research laboratories.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar - Solidifying Agent (Difco)</td>
			<td>Becton, Dickinson and Company (BD)</td>
			<td>281230</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>Mettler Toledo</td>
			<td>JL602-G/L</td>
			<td>Equipment&#160;</td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>Mettler Toledo</td>
			<td>AB54-S</td>
			<td>Equipment&#160;</td>
		</tr>
		<tr>
			<td>Antibiotic supplements cefoperazone, cycloheximide, trimethoprim, vancomycin</td>
			<td>Oxoid Ltd.</td>
			<td>SR0183E</td>
			<td></td>
		</tr>
		<tr>
			<td>Atmosphere Control Gas Pak&#160; (Campy, 85% N2, 10% CO2, and 5% O2)</td>
			<td>Becton, Dickinson and Company (BD)</td>
			<td>260680</td>
			<td></td>
		</tr>
		<tr>
			<td>Atmosphere Control Vessel,&#160; GasPak EZ CampyPak container system&#160;</td>
			<td>Becton, Dickinson and Company (BD)</td>
			<td>260678</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave - Amsco Lab250, Laboratory Steam Sterilizer</td>
			<td>Steris plc</td>
			<td>LV-250</td>
			<td>Equipment&#160;</td>
		</tr>
		<tr>
			<td>Biological Safety Cabinet, Type A2, Purifier Logic+</td>
			<td>Labconco Corporation</td>
			<td>302411101</td>
			<td>Equipment&#160;</td>
		</tr>
		<tr>
			<td>Bolton broth&#160;</td>
			<td>Oxoid Ltd.</td>
			<td>CM0983</td>
			<td></td>
		</tr>
		<tr>
			<td>Brucella broth (Difco)</td>
			<td>Becton, Dickinson and Company (BD)</td>
			<td>211088</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffered Peptone Water</td>
			<td>Bio-Rad Laboratories Inc.</td>
			<td>3564684</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Microcentrifuge 5424</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Centrifuge, Avanti J-25</td>
			<td>Beckman Coulter, Inc.&#160;</td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Chicken thighs, wings, drumsticks, livers</td>
			<td>Local retailers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Extraction - PreMan Ultra Sample Preparation Reagent&#160;</td>
			<td>Thermo Fisher Scientific Inc.&#160;</td>
			<td>4318930</td>
			<td></td>
		</tr>
		<tr>
			<td>FAM probe for hipO (Sequence 5&#39;-TTGCAACCTCACTAGCAAAATCC 
			ACAGCT-3&#39;)</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter - 0.45 &#181;m sterile cellulose acetate filter&#160;</td>
			<td>Merck-Millipore LTD</td>
			<td>DAWP04700</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter - 0.65 &#181;m sterile cellulose acetate filter&#160;</td>
			<td>Merck-Millipore LTD</td>
			<td>HAWG04700</td>
			<td></td>
		</tr>
		<tr>
			<td>forward primer for cdtA (Sequence 5&#39;-TGTCAAACAAAAAACACCAAGCT 
			T-3&#39; &#39;)</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>forward primer for hipO (Sequence 5&#39;-TCCAAAATCCTCACTTGCCATT-3&#39;)</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>forward primer for IAC (Sequence 5&#39;-GGCGCGCCTAACACATCT-3&#39;)</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEX probe for cdtA (Sequence 5&#39;-AAAATTTCCCGCCATACCACTTG 
			TCCC-3&#39;)</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator - Inova 4230 refrigerated incubator shaker</td>
			<td>New Brunswick Scientific</td>
			<td>4230</td>
			<td>Equipment&#160;</td>
		</tr>
		<tr>
			<td>Inoculating Loop - Combi Loop&#160; 10&#181;L and 1&#181;L&#160;</td>
			<td>Fisher Scientific International, Inc</td>
			<td>22-363-602</td>
			<td></td>
		</tr>
		<tr>
			<td>Internal Amplification Control (IAC) DNA fragment (Sequence 5&#39;-TGGAAGCAATGCCAAATGTGTAT 
			GTGGTGGCATTGTCTTCTCCC 
			GTTGTAACTATCCACTGAGATG 
			TGTTAGGCGCGCC-3&#39;)</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laked horse blood&#160;</td>
			<td>Remel Inc.</td>
			<td>R54072</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual pipette Pipet-Lite LTS Pipette L-1000XLS+</td>
			<td>Mettler Toledo</td>
			<td>17014382</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Manual pipette Pipet-Lite LTS Pipette L-100XLS+</td>
			<td>Mettler Toledo</td>
			<td>17014384</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Manual pipette Pipet-Lite LTS Pipette L-10XLS+</td>
			<td>Mettler Toledo</td>
			<td>17014388</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Manual pipette Pipet-Lite LTS Pipette L-200XLS+</td>
			<td>Mettler Toledo</td>
			<td>17014391</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Manual pipette Pipet-Lite LTS Pipette L-20XLS+</td>
			<td>Mettler Toledo</td>
			<td>17014392</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Manual pipette Pipet-Lite Multi Pipette L8-200XLS+</td>
			<td>Mettler Toledo</td>
			<td>17013805</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Manual pipette Pipet-Lite Multi Pipette L8-20XLS+</td>
			<td>Mettler Toledo</td>
			<td>17013803</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Media Storage Bottle -PYREX 1 L Square Glass&#160; Bottle, with GL45 Screw Cap</td>
			<td>Corning Inc.</td>
			<td>1396-1L</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Media Storage Bottle -PYREX 2 L Round Wide Mouth Bottle, with GLS80 Screw Cap</td>
			<td>Corning Inc.</td>
			<td>1397-2L</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Microtiter plate, 96 well plate, flat bottom, polystyrene, 0.34 cm2, sterile, 108/cs</td>
			<td>MilliporeSigma</td>
			<td>Z707902</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixer - Vortex Genie 2</td>
			<td>Scientific Industries Inc.</td>
			<td>SI-0236</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Motorized pipette controller, PIPETBOY2</td>
			<td>INTEGRA Biosciences Corp.</td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>PCR Mastermix 2&#215; TaqMan Gene Expression&#160;</td>
			<td>Thermo Fisher Scientific Inc.&#160;</td>
			<td>4369542</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish Rotator -&#160; bioWORLD Inoculation Turntable</td>
			<td>Fisher Scientific International, Inc</td>
			<td>3489E20</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Petri Dishes with Clear Lid (100 mm x 15mm)</td>
			<td>Fisher Scientific International, Inc</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips GP LTS 1000&#181;L S 768A/8</td>
			<td>Mettler Toledo</td>
			<td>&#160;30389273</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips GP LTS 20&#181;L 960A/10</td>
			<td>Mettler Toledo</td>
			<td>30389270</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips GP LTS 200&#181;L F 960A/10</td>
			<td>Mettler Toledo</td>
			<td>30389276</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent Reservoir, 25 mL sterile reservoir used with multichannel pipettors</td>
			<td>Thermo Fisher Scientific Inc.&#160;</td>
			<td>8093-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Realtime PCR - 7500 Real-Time PCR system&#160;</td>
			<td>(Applied Biosystems, Foster City, CA)</td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>reverse primer for cdtA (Sequence 5&#39;-CCTTTGACGGCATTATCTCCTT-3&#39;)</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>reverse primer for hipO (Sequence 5&#39;-TGCACCAGTGACTATGAATAACG 
			A-3&#39;)</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>reverse primer for IAC (Sequence 5&#39;-TGGAAGCAATGCCAAATGTGTA 
			-3&#39;)</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes, Nunc Serological Pipettes (10 mL)</td>
			<td>Thermo Fisher Scientific Inc.&#160;</td>
			<td>170356N</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes, Nunc Serological Pipettes (2 mL)</td>
			<td>Thermo Fisher Scientific Inc.&#160;</td>
			<td>170372N</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes, Nunc Serological Pipettes (25 mL)</td>
			<td>Thermo Fisher Scientific Inc.&#160;</td>
			<td>170357N</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes, Nunc Serological Pipettes (50 mL)</td>
			<td>Thermo Fisher Scientific Inc.&#160;</td>
			<td>170376N</td>
			<td></td>
		</tr>
		<tr>
			<td>Spreader - Fisherbrand L-Shaped Cell Spreaders</td>
			<td>Fisher Scientific International, Inc</td>
			<td>14-665-230</td>
			<td></td>
		</tr>
		<tr>
			<td>Stomacher bag, Nasco Whirl-Pak Write-On Homogenizer Blender Filter Bags</td>
			<td>Thermo Fisher Scientific Inc.&#160;</td>
			<td>01-812</td>
			<td></td>
		</tr>
		<tr>
			<td>TAMRA probe for IAC (Sequence 5&#39;-TTACAACGGGAGAAGACAATGCC 
			ACCA-3&#39;)</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler (GeneAmp PCR system 9700)</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Water Filtration - Elga Veolia Purelab Flex&#160;</td>
			<td>Elga LabWater</td>
			<td>PF2XXXXM1-US</td>
			<td>Equipment</td>
		</tr>
	</tbody>
</table>

detection and isolation of campylobacter spp. from raw meat

This article presents a rapid yet robust protocol for isolating Campylobacter spp. from raw meats, specifically focusing on Campylobacter jejuni and Campylobacter coli. The protocol builds upon established methods, ensuring compatibility with the prevailing techniques employed by regulatory bodies such as the Food and Drug Administration (FDA) and the U.S. Department of Agriculture (USDA) in the USA, as well as the International Organization for Standardization (ISO) in Europe. Central to this protocol is collecting a rinsate, which is concentrated and resuspended in Bolton Broth media containing horse blood. This medium has been proven to facilitate the recovery of stressed Campylobacter cells and reduce the required enrichment duration by 50%. The enriched samples are then transferred onto nitrocellulose membranes on brucella plates. To improve the sensitivity and specificity of the method, 0.45 &#181;m and 0.65 &#181;m pore-size filter membranes were evaluated. Data revealed a 29-fold increase in cell recovery with the 0.65 &#181;m pore-size filter compared to the 0.45 &#181;m pore-size without impacting specificity. The highly motile characteristics of Campylobacter allow cells to actively move through the membrane filters towards the agar medium, which enables effective isolation of pure Campylobacter colonies. The protocol incorporates multiplex quantitative real-time polymerase chain reaction (mqPCR) assay to identify the isolates at the species level. This molecular technique offers a reliable and efficient means of species identification. Investigations conducted over the past twelve years involving retail meats have demonstrated the ability of this method to enhance recovery of Campylobacter from naturally contaminated meat samples compared to current reference methods. Furthermore, this protocol boasts reduced preparation and processing time. As a result, it presents a promising alternative for the efficient recovery of Campylobacter from meat. Moreover, this procedure can be seamlessly integrated with DNA-based methods, facilitating rapid screening of positive samples alongside comprehensive whole-genome sequencing analysis.

Effect of moisture in Brucella agar plates for passive filtration of Campylobacter 
Campylobacter has a small genome and lacks several stress response genes commonly occurring in other bacteria, such as E. coli O157:H7 and Salmonella. Therefore, it is more sensitive to various environmental stresses and cannot tolerate dehydration or ambient oxygen levels. Conversely, an overly moist agar medium can flood the filter. This not only causes diffusion of the sample to outside the filter, but also increases exposure time to oxygen21.
To determine the appropriate conditions for filter-based isolation of Campylobacter, Brucella agar plates were dried with the lids removed for 0 h, 1 h, 2 h, and 3 h inside a biological safety cabinet and assessed for the efficiency of Campylobacter cells to traverse a 0.65 &#181;m pore-size filter. Four 20 &#181;L aliquots of C. jejuni S27 cultures at the concentrations of 1.53 x 104 and 1.53 x 105 CFU/mL were pipetted onto each filter membrane that had been placed on top of a Brucella plate. After 15 min of penetration, filters were removed, and plates were incubated overnight for cell growth.
Cells from 5 replicated plates were then counted and noted in Table 1. The results indicated that the agar plates dried for 2 h and 3 h performed similarly with nearly equal numbers of cells recovered from passive filtration. Noticeably, the plates dried under these conditions for 0 h and 1 h did not allow cells to fully traverse the membrane within the 15 min time period used.
Comparison of different pore-size filter membranes for isolating Campylobacter from chicken livers 
Considering the Campylobacter cell sizes (0.5-5 &#181;m in length and 0.2-0.9 &#181;m in width) and a wide range of food particle sizes, cellulose acetate filters with 0.45 &#181;m and 0.65 &#181;m pore sizes were tested for the efficiency of Campylobacter passage when given a 15 min incubation time. Food samples consisting of 450 g of chicken livers spiked with 153 CFU of C. jejuni and then enriched overnight were used for the experiment. As a no-filter control, direct plating of the enrichment sample was included in parallel. The results (Figure 2) from 5 replicate plates consistently showed that the 0.65 &#181;m pore size filter allowed more cells to traverse than the 0.45 &#181;m pore-size filters, resulting in increases of ~29-fold more cells obtained. The 0.45 &#181;m pore size filter retained too many cells on the upper side of the filter, resulting in a significantly lower recovery of Campylobacter from food compared to the 0.65 &#181;m pore size filter. As expected, there was a lawn of different background organisms growing on the no-filter control plates.
Application of passive filtration in Campylobacter isolation from retail chicken 
Because of the unusual motility of Campylobacter cells, the passive filtration technique was selected for the isolation of C. jejuni and C. coli from retail meat products, which are typically contaminated with numerous background organisms. Between the years 2014-2023, a total of 79 raw meat packages, including different parts of chicken meat, chicken livers, beef livers, and calf livers, were collected from various local supermarkets. From each package, 450 g was sampled for the isolation of Campylobacter spp. By combining selective enrichment of Campylobacter in blood-containing Bolton Broth and passive filtration of the cells through a 0.65 &#181;m pore size cellulose acetate filter directly onto a Brucella agar plate, 49 Campylobacter strains have been successfully isolated from 79 meat samples (Table 2). Figure 3 represents the result of isolating a new Campylobacter strain from chicken livers. The method has been repeatedly proven to be sensitive, specific, and cost-effective.
Identification and differentiation of C. jejuni and C. coli isolates 
To verify the genus and differentiate the species of Campylobacter isolates obtained from raw meat, a multiplex qPCR assay amplifying the specific gene targets (hipO and cdtA) for C. jejuni and C. coli, and an internal amplification control (IAC) was employed. The IAC was included as a false-negative indicator in the concurrent amplification of multiple genes. The assay was implemented in a 96-well format with rapid cell lysis and DNA extraction using a commercially available reagent (see Table of Materials). Table 2 summarizes the result of species identification of the C. jejuni and C. coli strains. As additional verification, whole-genome sequencing results confirmed the species of all the isolates (data not shown).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66462/66462fig01.jpg" /> 
Figure 1: A workflow diagram for the isolation and identification of Campylobacter species from retailed meat. <a href="https://www.jove.com/files/ftp_upload/66462/66462fig01large.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/66462/66462fig02.jpg" /> 
Figure 2: The impact of filter size on recovery of Campylobacter. The results indicate that the 0.65 &#181;m filter can recover an average of 900 &#177; 138 colonies, while the 0.45 &#181;m filter recovers 31 &#177; 7 colonies. The results were generated from N = 20 (5 plates with 4 drops/plate) and a Student&#39;s t-test indicates the means are statistically different (p &#60; 0.0001). <a href="https://www.jove.com/files/ftp_upload/66462/66462fig02large.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/66462/66462fig03.jpg" /> 
Figure 3: Isolation of Campylobacter by passive filtration of enriched poultry samples. (A) Depicts the four 20 &#181;L drops of enriched sample deposited on the nitrocellulose membrane filter. The image was collected during the 15 min of passive filtration. (B) Depicts the plate after the nitrocellulose filter was removed. The four spots indicate where the enriched sample traversed the membrane. (C) Depicts the plate following 24 h incubation. <a href="https://www.jove.com/files/ftp_upload/66462/66462fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: The effect of the drying time of Brucella agar plates on the passive filtration of C. jejuni. <a href="https://www.jove.com/files/ftp_upload/66462/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: C. jejuni and C. coli strainsisolated from raw meat. During the period of time ranging from 2008 to 2023, 36 C. jejuni and 13 C. coli strains were isolated from 79 meat packages across 24 unique retail products. <a href="https://www.jove.com/files/ftp_upload/66462/Table 2.xlsx" target="_blank">Please click here to download this Table.</a>
Supplemental File 1: Images throughout the isolation and enumeration processes. <a href="https://www.jove.com/files/ftp_upload/66462/Supplemental File 1.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Author Spotlight: Development of an Enhanced Protocol for Rapid and Accurate Isolation of Campylobacter  from Food Products at JoVE.com

Author Spotlight: Development of an Enhanced Protocol for Rapid and Accurate Isolation of Campylobacter from Food Products

Campylobacter is the leading cause of bacterial foodborne gastroenteritis worldwide. Despite establishments enacting measures to reduce the prevalence throughout their facilities, contaminated products consistently reach consumers. The technique developed over the past twelve years addresses limitations of existing methods for isolating and detecting Campylobacter spp. from raw meat.

Detection and Isolation of Campylobacter spp. from Raw Meat

This article presents a rapid yet robust protocol for isolating Campylobacter spp. from raw meats, specifically focusing on Campylobacter jejuni and ...

detection-and-isolation-of-campylobacter-spp-from-raw-meat

Research

JoVE Journal

Biology

1.6K Views.  Agricultural Research Service (ARS), United States Department of Agriculture (USDA). Campylobacter is the leading cause of bacterial foodborne gastroenteritis worldwide. Despite establishments enacting measures to reduce the prevalence throughout their facilities, contaminated products consistently reach consumers. The technique developed over the past twelve years addresses limitations of existing methods for isolating and detecting Campylobacter spp. from raw meat.

Video: Author Spotlight: Development of an Enhanced Protocol for Rapid and Accurate Isolation of Campylobacter  from Food Products

Isolation of Genomic DNA from Mouse Tails

Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable cytokine-secreting cells. In order to label IL-17-secreting cells, a single cell suspension of mouse splenocytes is prepared and stimulated at 37&deg;C with PMA/ionomycin to induce cytokine secretion. To stop secretion cells are then placed on ice and are exposed to the IL-17 Catch Reagent   a bi-specific antibody that binds to CD45 on the cell surface of leukocytes and to IL-17 as it is secreted and caught near the cell surface. Secretion is then re-started by increasing the temperature to 37&deg;C and IL-17 is trapped by the Catch Reagent. Secretion is then stopped again, by placing cells on ice. To detect the trapped IL-17, cells are incubated with a second IL-17-specific antibody conjugated to biotin and an Anti-Biotin-PE antibody. Cells can now be directly analyzed by flow cytometry or prepared for isolation and enrichment by subsequent labeling with Anti-PE conjugated MicroBeads.

This procedure describes the detection and isolation of mouse TH17 leukocytes that actively secrete IL-17 upon stimulation.

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable ...

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...