Development of this protocol was supported by an operating grant from CIHR (MOP97807) to SED.

Safety Statement
This protocol describes infection of mice with live L. monocytogenes. The pathogen is handled safely under BSL2 conditions by trained personnel who are not immunocompromised. Immunocompromised people include pregnant women, the elderly and individuals who are HIV-infected or have chronic conditions that require treatment with immunosuppressive therapy. Personnel should don a protective lab coat or gown, gloves, mask, and eye protection while handling infected samples. T...

No conflicts of interest to disclose.

Here we describe a protocol of how to carry out a basic experimental infection with the EGD strain of L. monocytogenes25 in male or female C57BL/6J mice. This protocol was set up for the purpose of studying the effect of a novel small molecule IS001 on IFN-&#947; production by innate and adaptive lymphocytes in vivo31. By monitoring bacterial clearance and survival post-infection, insights were gained into how these changes in IFN-&#947; impacted the host&#39;s ability to control t...

IFN-&#947; is a cytokine that is crucial for mediating immunity against intracellular pathogens and for controlling tumor growth5. The importance of this cytokine in bacterial resistance is evident in the observation that humans with mutations in the IFN-&#947; signaling pathway are highly susceptible to infection with mycobacteria and salmonellae6. Similarly, mice deficient in either IFN-&#947; or the IFN-&#947; receptor exhibit defects in resistance to mycobacteria7-9 and other intracellular pathogens including L. monocytogenes10,11, Leishmania major12, Salmonella typhimurium13, and certain viruses11. In addition to combatting pathogens, IFN-&#947; plays a crucial role in host-defense against tumors14. Though higher production of IFN-&#947; is beneficial in the context of infection or cancer, prolonged production of this cytokine has been linked to the development of systemic autoimmunity15-17 and the acceleration of type I diabetes in the non-obese diabetic mouse model18.
The major sources of IFN-&#947; include NK cells, NKT cells, &#947;&#948; T cells, T helper 1 (Th1) cells, and cytotoxic T lymphocytes (CTL)5,19,20. IFN-&#947; enhances both innate and adaptive immunity by: (1) up-regulating major histocompatibility complex (MHC) class I and II expression, (2) increasing the expression of co-stimulatory molecules on antigen presenting cells, (3) enhancing macrophage phagocytosis and the production of pro-inflammatory cytokines and microbicidal factors (e.g., nitric oxide and reactive oxygen species), (4) promoting the differentiation of na&#239;ve CD4+ T cells into Th1 effector cells, (5) promoting antibody class switching to immunoglobulin (Ig)2a and IgG3 (in mouse), (6) inducing the production of chemokines to recruit immune cells to sites of infection, and (7) enhancing NK cell and CTL responses5,19. Given the crucial importance of IFN-&#947; in the host response to pathogens and tumors, recombinant IFN-&#947; has been tested as a treatment for various infections and malignancies (reviewed in19). However, because systemic administration of IFN-&#947; or the Th1 promoting cytokine interleukin-12 (IL-12) is associated with side effects and dose-related toxicity19,21, there is interest in developing alternative strategies to increase IFN-&#947; production by immune cells. Development of new biologics and small molecules requires in vivo screening tools to test whether such agents increase IFN-&#947; production during an immune response and whether this translates into meaningful biological effects such as increases in animal survival.
Experimental infection of mice with the gram-positive bacterium L. monocytogenes has been an instrumental model for deciphering the role of IFN-&#947; in host-immunity against intracellular pathogens1,22. Infection of mice with the pathogen intravenously or intraperitoneally (i.p.) leads to the rapid dissemination of the bacteria to the spleen and liver, where they become internalized by resident macrophages and hepatocytes with peak bacterial loads in the spleen occurring between 3 and 4 days post-infection1,3,22. Production of IFN-&#947; by NK cells is important for macrophage activation and early resistance against the pathogen3; however at high infectious doses, production of IFN-&#947; can also be detrimental to pathogen clearance23. NKT cells are also a source of IFN-&#947; in the spleen and liver during early control of pathogens2,24 and this production has been shown to amplify IFN-&#947; production by other cell types including NK cells2. On the other hand, later-acting adaptive T lymphocytes, CD8+ T cells in particular, are important for mediating the clearance of the pathogen and providing protection against re-infection1,4,22.
This infection model has been attractive to researchers for a number of reasons (reviewed in1). First, infection with the pathogen is highly reproducible and induces a strong Th1 and cellular immune response. Secondly, during sublethal infection, bacterial load is concentrated in the liver and spleen where it can be easily measured. Thirdly, the pathogen can be safely handled under Biosafety Level 2 (BSL2) conditions. Fourthly, the organism and the immune response that it generates have been extensively characterized. Finally, a variety of mutant and genetically-modified strains have been developed that are available for use.
Described here is a basic protocol for inoculation of C57BL/6J mice with the EGD strain of L. monocytogenes25 and for measuring IFN-&#947; responses by NK, NKT, and adaptive lymphocytes post-infection. Also described is how to measure bacterial load in the spleen and liver after sublethal infection and to carry out survival studies after infection with a modified LD50 dose of the pathogen. Finally, representative data are shown of how this protocol can be used to screen the effect of new treatments on IFN-&#947; responses and mouse survival from L. monocytogenes infection.

<table border="0" cellpadding="0" cellspacing="0" width="923">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Brain Heart Infusion Broth, Modified</td>
			<td style="width:191px;">BD</td>
			<td style="width:107px;">299070</td>
			<td style="width:273px;">any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>BD</td>
			<td style="width:107px;">214010</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Triton X-100</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:107px;">X100</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">1xPBS</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:107px;">D8537</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">TissueLyser II</td>
			<td style="width:191px;">Qiagen</td>
			<td style="width:107px;">85300</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Ammonium Chloride (NH3Cl)</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">KHCO3</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Na2EDTA</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">RPMI 1640</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:107px;">22400089</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Fetal Bovine Serum&#160;</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:107px;">12483</td>
			<td>Before use, heat-inactivate at 56 &#176;C for 30 min</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">L-glutamine</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:107px;">25030</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Non-essential amino acids</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:107px;">11140</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Penicillin/Streptomycin</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:107px;">15140</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">GolgiStop Protein Transport Inhibitor (containing Monensin)</td>
			<td style="width:191px;">BD</td>
			<td style="width:107px;">554724</td>
			<td>Use 4 &#956;l in 6 ml cell culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">16% Paraformaledehye</td>
			<td style="width:191px;">Electron Microscopy Sciences</td>
			<td style="width:107px;">15710</td>
			<td>Dilute to 4% PFA in ddH20 or 1xPBS</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:353px;">10 x Perm/Wash buffer</td>
			<td style="width:191px;">BD</td>
			<td style="width:107px;">554723</td>
			<td>Dilute 10x in ddH20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Fc block, Anti-Mouse CD16/CD32 Purified</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">14-0161</td>
			<td>Dilute 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Fixable Viability Dye eFluor 506</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">65-0866</td>
			<td>Dilute 1:1000 (we have also used viability dyes from Molecular Probes)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">anti-Mouse CD4-PE-Cy5 (GK1.5)</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">15-0041</td>
			<td>Manufacturer recommends a certain test size; however this should be titrated before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">anti-Mouse CD8-FITC (53-6.7)</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">11-0081</td>
			<td>Manufacturer recommends a certain test size; however this should be titrated before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">PBS57/mCD1d tetramer-APC</td>
			<td style="width:191px;">NIH Tetramer Core Facility</td>
			<td style="width:107px;">N/A</td>
			<td>Obtained as a gift from the facility</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Mouse TCR&#946;-PE-Cy7 (H56-597)</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">25-5961</td>
			<td>Manufacturer recommends a certain test size; however this should be titrated before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Mouse NKp46-APC-eFluor780 (29A1.4)</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">47-3351</td>
			<td>Manufacturer recommends a certain test size; however this should be titrated before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">anti-Mouse CD45 PE-Cyanine7 (30-F11)</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">25-0451</td>
			<td>Manufacturer recommends a certain test size; however this should be titrated before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">anti-Mouse IFN gamma-PE (XMG1.2)</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">12-7311</td>
			<td>Manufacturer recommends a certain test size; however this should be titrated before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">OneComp eBeads</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">01-1111</td>
			<td>Manufacturer recommends a certain test size; however this should be titrated before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Mouse IFN gamma ELISA kit</td>
			<td style="width:191px;">eBioscience</td>
			<td>88-7314</td>
			<td>Used for measuring the interferon gamma in the culture supernatant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">50 mL vented tubes for culture</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>Used for culturing the bacteria, any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">1.5 ml microcentrifuge tubes</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">bacterial petri dishes</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">2 ml cyrovials</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">UV spectrometer</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">safety engineered needles</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">C57BL6/J</td>
			<td style="width:191px;">Jackson laboratories</td>
			<td style="width:107px;">Stock#000664</td>
			<td>Order for arrival at 7 wks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Bleach</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>For decontamination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">70% Ethanol</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>For decontamination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Glass beads</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Centrifuge</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>rotor, buckets, bucket covers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Microcentrifuge</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Sterile Glycerol</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Pipette Tips</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Pipette</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Surgical instruments</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">70 micron strainers</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">3 ml syringe</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Pipette gun</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Filtration Units</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Trypan Blue</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>Dilute 1 to 9 in ddH20, any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Hemocytometer</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Round bottomed plates</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">FACs tubes</td>
			<td style="width:191px;">BD</td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">BD LSR II</td>
			<td style="width:191px;">BD</td>
			<td style="width:107px;"></td>
			<td>Any flow cytometer could be used for acquisition that has an appropriate laser configuration and filter set to discriminate the fluorochormes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Flowjo software</td>
			<td style="width:191px;">Treestar</td>
			<td style="width:107px;"></td>
			<td>Used for data analysis. Other types of data analysis software will also be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Multichannel pipettor (0-300 &#181;l)</td>
			<td style="width:191px;">Eppendorf</td>
			<td style="width:107px;"></td>
			<td>Used for washing cells and adding antibodies during flow cytometry staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Acetic Acid</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>Used for washing glass beads, any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Microbank Bacterial Preservation System</td>
			<td style="width:191px;">Pro-lab Diagnositics</td>
			<td style="width:107px;"></td>
			<td>Used as an alternative to glycerol stocks for long-term storage of bacteria</td>
		</tr>
	</tbody>
</table>

experimental infection with listeria monocytogenes as a model for studying host interferon-&#947; responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Figure 3 presents some typical flow cytometry staining of IFN-&#947; in splenic NK and NKT cells at 24 hr post-infection with 105 CFU of the pathogen. This figure also illustrates the gating strategy for the staining panel described in Table 2. Figure 4 shows some representative data that were obtained in one experiment where male mice were treated with the PPAR&#945; antagonist IS001 or vehicle control, infected with 105<...

Watch this Scientific Journal Video about Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-Î³ Responses at JoVE.com

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

The overall goals of this protocol are to cultivate and infect C57 blacksix mice with the EGG strain of listeria monocytogenes and to measure the bacterial load, interferon gamma responses, and survival and response to infection by this pathogen. This protocol can help researchers screen for agents or genes that have an impact on interferon gamma responses and on animal survival after bacterial infection. The main advantage of this technique is that it is relatively simple to set up in the laboratory.Even for those with little experience in cultivating bacteria. Also demonstrating the procedure will be Jennifer Ahn, a graduate student from my laboratory. Begin by dipping a sterile pipette tip into room temperature thawed glycerol stock of the L monocytogenes EGG strain.And immediately streak the tip back and forth across a section of a brain heart infusion or BHI plate. This is the primary streak. Turn the plate 90 degrees and drag a fresh pipette tip through the first streak, spreading the stock to the next quarter of the plate.This is the secondary streak. Then, turn and spread the secondary streak into a third quarter of the plate to make the tertiary streak. Now, turn the plate upside down at 37 degrees celsius, for an overnight incubation.Single uniform colonies should be visible in the last set of streaks between 16 and 24 hours. When the colonies appear, dispense 10 milliliters of sterile BHI broth into a sterile vented 50 milliliter tube and use a sterile pipette tip to transfer one L monocytogenes colony from the plate into the broth. Culture the inoculated broth in an orbital shaking incubator overnight at 37 degrees celsius and 225 rotations per minute, until the OD600 equals one.For preparation of the experimental L monocytogenes infection inoculum, in a biosafety cabinet, inoculate a tube of BHI medium with 100 microliters of the overnight culture. After another incubation at 37 degrees celsius, with shaking until the desired OD600 is reached, transfer the culture contents into a sterile centrifuge tube for centrifugation. And use a vacuum attached to a trap flask containing bleach to aspirate the supernatant.Wash the pellet two times with PBS under the same centrifuge conditions. After the second wash, resuspend the bacteria in PBS and dilute at the appropriate concentration. To deliver the CFU of interest to each mouse in a 200 microliter volume.Next, mix the suspension thoroughly with a sterile pipette tip to ensure that the bacteria are homogeneously distributed. Then draw 200 microliters of inoculum into a one milliliter safety engineered syringe fitted with a 25 gauge needle. Now, scruff a mouse with the less dominant hand by the loose skin around the shoulders.And inject all 200 microliters of the inoculum in the lower quadrant of the abdomen, just lateral to the midline to avoid the bladder, using a new needle and syringe for each animal. To measure the peak infection bacterial load, on day three post infection, restrain the limbs of an experimental animal in the supine position on a dissecting board. And disinfect the skin with 70 percent ethanol.Using sterile tough cut scissors, make an incision from the chest to the groin. Then make an incision from the mid groin toward each knee and from the mid chest toward each elbow. Blunt, dissect, and reflect back the skin, pinning it open with 25 gauge needles.Disinfect the muscle layer with more 70 percent ethanol. Then using sterile fine scissors, make a midline incision in the peritoneal wall. Use forceps to grasp the xiphoid process.Make incisions in the peritoneal wall starting at the xiphoid process and moving laterally on each side. Following the ribcage to just below the diaphragm. This will reveal the liver.Next, use sterile scissors to cut an approximately 100 milligram piece out of one lobe. Place the tissue in a preweighed 1.5 milliliter micro centrifuge tube containing 0.2 to 0.3 grams of 1.5 to 2 millimeter acid washed sterile glass beads. And use the forceps to gently push aside the organs on the left side of the peritoneal cavity to visualize the spleen.Use the forceps to gently grasp the spleen, cutting away the surrounding connective tissue to release the organ from the peritoneal cavity. Place the spleen in a different tube than the liver, and transfer the tissues to the laboratory in a leak proof container containing ice. Shake the tubes while using a bead mill homogenizer for three minutes at a 30 hertz frequency.Then, set up a tenfold dilution series of the homogenates in 0.1 percent trident X 100 in PBS. Next, use a sterile spreader to spread 100 microliters of each diluted homogenate onto a BHI agar plate. And transfer the plates to a 37 degrees celsius incubator for an overnight incubation.Keep the plates that contain up to 300 colonies per plate. Count the colonies on each plate. To measure CD4 in CT8 t cell interferon gamma responses, harvest the spleens from mice infected with two tenths into the fourths CFU of the pathogen as just demonstrated.Process the spleens into a single red blood cell life suspension. And resuspend the pellet at four times ten to the six cells per milliliter concentration in complete RPMI medium containing FCS. Next, seed one milliliter of cells per well into a 24 well plate with an equal number in volume of thawed, heat killed L monocytogenes and place the cold cultures into a 37 degrees celsius incubator.After 20 hours, add 0.66 microliters per milliliter of protein transport inhibitor to the wells. And return the plate to the incubator. Four hours later, in a biosafety cabinet, collect 500 microliters of culture supernatant and freeze the samples at negative 80 degrees celsius for the later measurement of interferon gamma by Eliza.Then transfer the cells into a single sterile 15 milliliter tube, washing the wells with PBS and pooling the wash with the collected cells for flow cytometric staining and analysis. The peak of bacterial load occurs in the spleen two to three days post infection. And is reduced dramatically by the addition of a PPAR Alpha antagonist prior to infection.At seven days post infection, the restimulation of spleen CD4 positive and CD8 positive t cells x vivo with heat killed pathogen elicits a positive interferon gamma response from CD4 positive and CD8 positive cells as detected by flow citometry. Treatment with the PPAR alpha antagonist prior to infection boosts the interferon gamma responses by CD4 and CD8 cells. Further, PPAR alpha antagonist treatment increases most survival after infection with the LD50 dose of the pathogen, illustrating the facility of this model for investigating the impact of new drugs that modulate interferon gamma responses on animal survival from infection.Once mastered, all the procedures in this protocol can generate the desired data in several weeks, if performed properly. The technique can be further adapted by infecting mice with strains of listeria monocytogenes that express model antigens and using MAC class one and two tetramery agents specific for these antigens. This will allow one to measure T cell expansion post infection.After its development, this technique became an instrumental model for deciphering the role of interferon gamma in host immunity to intracellular pathogens. After watching this video, you should have a good understanding of how to cultivate and infect C57 blacksix mice with listeria monocytogenes. And to measure the backtrail load and interferon gamma responses to infection.Don't forget that working with listeria monocytogenes can be hazardous to immuno compromised individuals such as pregnant women, the very young or elderly. Therefore the pathogen should only be handled by immuno competent individuals under biosafety level two conditions.

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Research

JoVE Journal

Immunology and Infection

13.4K vues.  University of Toronto. The overall goals of this protocol are to cultivate and infect C57 blacksix mice with the EGG strain of listeria monocytogenes and to measure the bacterial load, interferon gamma responses, and survival and response to infection by this pathogen. This protocol can help researchers screen for agents or genes that have an impact on interferon gamma responses and on animal survival after bacterial infection. The main advantage of this technique is that it is relatively simple to set up in the la...