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

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 the infection.
Critical Considerations in the Protocol
An important consideration in the design of this type of study is that each experiment be adequately powered and appropriately controlled. Due to biological variation in the immune response to infection (see Figures 4 and 6), it is recommended that N = 4 - 5 mice per group should be used for the initial immune studies. If after these studies there is a trend in the data, but no significant difference apparent between groups, a power calculation could be done to determine the least number of animals required in subsequent studies to achieve statistical significance. Regarding controls, it is important to include uninfected controls for determination of baseline IFN-&#947; responses for immune studies and vehicle controls to help distinguish the effect of the treatment from the stress associated with administering the treatment. Another important consideration is the timing of treatment. Since the innate response to L. monocytogenes is very rapid, it is recommended that the first treatment be administered on the day prior to, or at the same time as, inoculation in order to ensure that therapeutic levels of the reagent be achieved prior to the initiation of the innate immune response.
Yet another important consideration is the dose of the pathogen to be used for infection. A sublethal dose is recommended for measurement of bacterial load, since it increases the chance that the pathogen will be concentrated within the spleen and liver, allowing for the more accurate enumeration of the bacteria. A sublethal dose is also recommended for enumerating IFN-&#947; responses by adaptive lymphocytes to ensure that animals do not succumb to listeriosis prior to the time of peak T cell expansion. In contrast, it is recommended that a higher infectious dose be used for measurement of the early NK and NKT cell response at 24 hr in order to maximize the IFN-&#947; production by these cells.
The classical LD50 is the dose of pathogen that results in 50% lethality of mice. Since death was not an acceptable endpoint at our institution and since many symptoms of listeriosis can predict whether an animal is likely to succumb to an infection, we used a defined list of clinical signs instead of death as an endpoint in our studies. Using this method, it was determined that the modified LD50 was 105 CFU for 8-week-old male and 1.5 x 105 CFU for 8-week-old female C57BL/6J mice31. These LD50 doses were determined by measuring the percent survival of mice to endpoints in step-wise dose-escalation studies (N = 5 studies in total) that each contained N = 8 mice per group (e.g., mice were infected first with 10,000 CFU, then a second batch with 20,000 CFU, etc.). The LD50 calculation was determined from a regression plot of the log (CFU) (x-axis) versus the probit of the percent survival values (y-axis) (website: userwww.sfsu.edu/efc/classes/biol710/probit/ProbitAnalysis.pdf).
Note that the modified LD50 dose determined in our lab may differ from that in another lab even when infecting mice with the same strain of L. monocytogenes. Part of this variability may relate to the subjective nature of monitoring clinical signs of listeriosis compared to the more absolute endpoint of death. Additional variability can result from differences in environmental factors such as mouse diet or the microbiota or differences in the preparation of inoculum between labs. Thus, it is recommended that prior to embarking on any survival studies, a pilot study be performed where female mice (N = 8 mice/group) are infected with 1.5 x 105 CFU of the same strain of L. monocytogenes as used in this study and symptoms monitored to determine if this dose indeed results in 50% survival to endpoints. If survival is lower or higher than 50% at this CFU, step-wise dose escalation or dose de-escalation studies could be performed to quickly narrow in on the LD50 dose.
Another important consideration is the strain or substrain of mice used for infection studies. This protocol describes infection of the commonly-used inbred mouse strain C57BL/6J. This strain is well-suited for measurement of IFN-&#947; responses since this mouse is considered to be a Th1-prone strain33 and as a result, is relatively resistant to L. monocytogenes infection (compared to Th2-prone mouse strains such as BALB/c)34,35. Adapting this protocol to other mouse strains will require knowledge of the infectious dose of the pathogen for the particular strain. It is also recommended to use mice of the same age, sex and vendor as outlined in this protocol in order to reduce the amount of trouble-shooting involved in setting up the model. For example, C57BL/6 mice ordered from one vendor (e.g., C57BL/6J) can exhibit genetic differences than C67BL/6 mice ordered from another vendor (e.g., C57BL/6NTac)36. In addition, the intestinal microbiota differs between C57BL/6 substrains obtained from different vendors, which can influence the balance of Th1 and Th17 responses in the mouse37.
Potential Modifications to Technique
Mice are most commonly inoculated i.p. or intravenously as opposed to the natural route of infection in humans, which is through the gastrointestinal tract. Oral infections are less common because standard strains of L. monocytogenes inefficiently infect the intestinal epithelium of mice38. This is because there is a single amino acid change in the sequence of mouse E-cadherin from human E-cadherin that results in loss of recognition of E-cadherin by the listerial invasion protein, internalin A (InIA)39. To overcome this barrier, researchers use mice that are transgenic for human E-cadherin protein or use listeria that have been engineered to express a mutated sequence of InIA (InIAmut) that binds to mouse E-cadherin with the same affinity as WT EGD for human E-cadherin40. Thus, one potential modification of this technique is to infect mice via the oral route. The reader is referred to another JoVE publication that describes oral inoculation methods38. Note that altering the mode of infection will affect the infectious dose as well as the kinetics of dissemination of the pathogen.
This protocol describes using heat-killed listeria to elicit IFN-&#947; production by CD4+ and CD8+ T cells. Heat-killed L. monocytogenes was chosen as a stimulus in our studies, because this antigen is inexpensive and because our lab was primarily interested in CD4+ T cell responses to the pathogen. One limitation is that heat-killed bacteria do not efficiently prime CD8+ T cell responses either in vitro41 or in vivo42,43 infection. Thus, the CD8+ T cell IFN-&#947; production that we observed by splenocytes harvested at the peak of infection (i.e., Figure 6) likely is in response to the residual live bacteria present in the splenocyte cultures or was elicited as a result of cytokine-induced cytokine release41. As an alternative to heat-killed listeria, one could also elicit IFN-&#947; responses ex vivo by exposing T cells to peptides encoding epitopes on listerial proteins. Indeed, immunodominant MHC Class II-restricted epitopes for listeriolysin O and the p60 hydrolase and have been described for C57BL/6 and BALB/C mice and immunodominant MHC Class I epitopes have been described for BALB/c44. Yet another approach is to infect mice with strains of L. monocytogenes that have been engineered to express model antigens such as ovalbumin or viral antigens in order to take advantage of existing MHC Class I- and MHC Class II-tetramer reagents to enumerate antigen-specific T cells in infected mice45,46.
Other Limitations of the Protocol
Another limitation of this model is that it only measures IFN-&#947; production by immune cells in the spleen. In addition to the use of tetramers to enumerate antigen specific T cells (of ovalbumin-expressing variants of L. monocytogenes), flow cytometry staining panels described here could be easily modified to measure the production of other cytokines such as TNF or IL-2 or effector molecules that participate in CD8 T cell or NK-mediated killing of the pathogen such as perforin or granzyme B. In addition, this protocol could also be adapted to examine IFN-&#947; produced by immune cells in the liver.
Future Applications
Once this protocol is mastered, it can serve as a simple in vivo model to screen the effects of various agents or genes on Th1 and cellular immunity.

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><tbody><tr><td>Brain Heart Infusion Broth, Modified</td><td>BD</td><td>299070</td><td>any brand should be appropriate</td></tr><tr><td>Agar</td><td>BD</td><td>214010</td><td>any brand should be appropriate</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td>any brand should be appropriate</td></tr><tr><td>1x PBS</td><td>Sigma</td><td>D8537</td><td>any brand should be appropriate</td></tr><tr><td>TissueLyser II</td><td>Qiagen</td><td>85300</td><td>any brand should be appropriate</td></tr><tr><td>Ammonium Chloride (NH&lt;sub&gt;3&lt;/sub&gt;Cl)</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>KHCO&lt;sub&gt;3&lt;/sub&gt;</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;EDTA</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>22400089</td><td>any brand should be appropriate</td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Gibco</td><td>12483</td><td>Before use, heat-inactivate at 56 &amp;deg;C for 30 min</td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030</td><td>any brand should be appropriate</td></tr><tr><td>Non-essential amino acids</td><td>Gibco</td><td>11140</td><td>any brand should be appropriate</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140</td><td>any brand should be appropriate</td></tr><tr><td>GolgiStop Protein Transport Inhibitor (containing Monensin)</td><td>BD</td><td>554724</td><td>Use 4 &amp;mu;l in 6 ml cell culture</td></tr><tr><td>16% Paraformaldehye</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Dilute to 4% PFA in ddH&lt;sub&gt;2&lt;/sub&gt;O or 1x PBS</td></tr><tr><td>10x Perm/Wash buffer</td><td>BD</td><td>554723</td><td>Dilute 1x in ddH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Fc block, Anti-Mouse CD16/CD32 Purified</td><td>eBioscience</td><td>14-0161</td><td>Dilute 1:50</td></tr><tr><td>Fixable Viability Dye eFluor 506</td><td>eBioscience</td><td>65-0866</td><td>Dilute 1:1,000 (we have also used viability dyes from Molecular Probes)</td></tr><tr><td>anti-Mouse CD4-PE-Cy5 (GK1.5)</td><td>eBioscience</td><td>15-0041</td><td>Manufacturer recommends a certain test size; however this should be titrated before use.</td></tr><tr><td>anti-Mouse CD8-FITC (53-6.7)</td><td>eBioscience</td><td>11-0081</td><td>Manufacturer recommends a certain test size; however this should be titrated before use.</td></tr><tr><td>PBS57/mCD1d tetramer-APC</td><td>NIH Tetramer Core Facility</td><td>N/A</td><td>Obtained as a gift from the facility</td></tr><tr><td>anti-Mouse TCR&amp;beta;-PE-Cy7 (H56-597)</td><td>eBioscience</td><td>25-5961</td><td>Manufacturer recommends a certain test size; however this should be titrated before use.</td></tr><tr><td>anti-Mouse NKp46-APC-eFluor780 (29A1.4)</td><td>eBioscience</td><td>47-3351</td><td>Manufacturer recommends a certain test size; however this should be titrated before use.</td></tr><tr><td>anti-Mouse CD45 PE-Cyanine7 (30-F11)</td><td>eBioscience</td><td>25-0451</td><td>Manufacturer recommends a certain test size; however this should be titrated before use.</td></tr><tr><td>anti-Mouse IFN gamma-PE (XMG1.2)</td><td>eBioscience</td><td>12-7311</td><td>Manufacturer recommends a certain test size; however this should be titrated before use.</td></tr><tr><td>OneComp eBeads</td><td>eBioscience</td><td>01-1111</td><td>Use one drop per stain</td></tr><tr><td>Mouse IFN gamma ELISA kit</td><td>eBioscience</td><td>88-7314</td><td>Used for measuring the interferon gamma in the culture supernatant</td></tr><tr><td>50 ml vented tubes for culture</td><td></td><td></td><td>Used for culturing the bacteria, any brand should be appropriate</td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>bacterial petri dishes</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>2 ml cyrovials</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>UV spectrometer</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>safety engineered needles</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>C57BL/6J</td><td>Jackson laboratories</td><td>Stock#000664</td><td>Order for arrival at 7 weeks</td></tr><tr><td>Bleach</td><td></td><td></td><td>For decontamination</td></tr><tr><td>70% Ethanol</td><td></td><td></td><td>For decontamination</td></tr><tr><td>Glass beads</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Centrifuge</td><td></td><td></td><td>rotor, buckets, bucket covers.</td></tr><tr><td>Microcentrifuge</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Sterile Glycerol</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Pipette Tips</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Pipette</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Surgical instruments</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>70 micron strainers</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>3 ml syringe</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Pipette gun</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Filtration Units</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Trypan Blue</td><td></td><td></td><td>Dilute 1 to 9 in ddH&lt;sub&gt;2&lt;/sub&gt;O, any brand should be appropriate</td></tr><tr><td>Hemocytometer</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Round bottomed plates</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>FACs tubes</td><td>Fisher Scientific</td><td>14-959-5</td><td></td></tr><tr><td>BD LSR II</td><td>BD</td><td></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><td>Flowjo software</td><td>Treestar</td><td></td><td>Used for data analysis. Other types of data analysis software will also be appropriate</td></tr><tr><td>Multichannel pipettor (0 - 300 &amp;micro;l)</td><td>Eppendorf</td><td></td><td>Used for washing cells and adding antibodies during flow cytometry staining</td></tr><tr><td>Acetic Acid</td><td></td><td></td><td>Used for washing glass beads, any brand should be appropriate</td></tr><tr><td>Microbank Bacterial Preservation System</td><td>Pro-lab Diagnostics</td><td></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 CFU L. monocytogenes, and then analyzed for IFN-&#947; in NK and NKT cells after 24 hr. This figure shows that treatment with IS001 boosted IFN-&#947; responses by NKT cells, but not NK cells after infection with the pathogen. Figure 5 shows representative staining for IFN-&#947; in splenic CD4+ and CD8+ T cells at 7 days post-infection after re-stimulation ex vivo with heat-killed pathogen. This figure also shows the gating strategy for the staining panel described in Table 3. Figure 6 shows representative data that were obtained in one experiment where male mice were treated daily with the PPAR&#945; antagonist IS001 or vehicle control, infected with a sublethal dose of L. monocytogenes, and analyzed at 7 days post-infection. This experiment shows that treatment with IS001 enhanced IFN-&#947; responses by both CD4+ and CD8+ lymphocytes. Figure 7 shows representative data from a study that investigated the effect of the PPAR&#945; antagonist IS001 on mouse survival to endpoints after infection with the modified LD50 dose of the pathogen. Plotted is the percent survival of mice against time post-infection. This figure shows that treatment with IS001 increased the survival of male mice to endpoints. Together these data illustrate how this model can be applied to investigate the effects of new drugs or treatments on IFN-&#947; responses in vivo and to explore how these immune changes impact animal survival from infection.
<img alt="Figure 1" src="/files/ftp_upload/54554/54554fig1.jpg" /> 
Figure 1. Dissecting the Spleens from Infected Mice. This series of photos shows how to dissect the spleen from a dead mouse. (a) Lie the mouse on its right side and spray down the skin with 70% ethanol. (b) Using aseptic or sterile forceps and tough-cut scissors, incise the skin just below the bottom of the rib cage. (c) Spray down the exposed muscle layer with 70% ethanol. The spleen should be visible underneath the muscle layer (open arrow head). (d) Using aseptic or sterile forceps and fine scissors, incise the muscle layer to reveal the spleen. (e) Gently grab the spleen with the forceps and use fine scissors to cut the spleen away from surrounding the connective tissue. (f) Place the spleen in a 15 ml conical tube containing sterile 1x PBS. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54554/54554fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54554/54554fig2.jpg" /> 
Figure 2. Counting Splenocytes using a Hemocytometer. (a) shows the central grid of the hemocytometer. (b) shows an enlarged view of the central grid that contains 25 large squares (that each contain 16 smaller squares). The five large squares used for counting are highlighted in grey (4 corner squares plus the center square in the central grid). (c) shows an enlarged view of one of the large grey squares. To determine the cell volume in 106/ml, first count all the viable cells within the five large grey squares. In the example shown, this count is 215. When counting, make sure to only count all of the clear (non-blue) cells, including those that are touching the double lines on the right and bottom of the grid. Do not count the cells touching the double lines on the left and top of the grid. Take the total five square count and divide it by 10 to obtain the number of cells in 106/ml. In the example, 215 divided by 10 is 21.5 x 106 cells/ml. Note that these calculations only work if you are counting 5 of the large squares as highlighted and dilute your cells 1:1 in trypan blue. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54554/54554fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54554/54554fig3.jpg" /> 
Figure 3. Gating Strategy for Detection of IFN-&#947; Production in NK and NKT cells. First gate on lymphocytes on FSC-A by SSC-A plot. Then gate on those events that are on the diagonal on the FSC-H/FSC-A plot. These are the singlets. Then gate on live (AmCyan-) and CD8- cells. Then plot the tetramer staining against TCR&#946;. The NKT cells are within the double positive population and the NK cells are within the double negative population. Gate on the double positive cells, and plot NKp46 versus FSC. Gate on the NKp46 negative population, which are the NKT cells (this gate can be set by finding the point of division in the two populations from the NK cell plot). The NK cells are the tetramer- TCR&#946;-NKp46+ population. Within NK and NKT cell gates, the IFN-&#947;+ cells in the PE channel are identified after setting a gate based on the FMO control. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54554/54554fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54554/54554fig4.jpg" /> 
Figure 4. Representative Data Obtained for the Frequencies of IFN-&#947;+ NK and NKT Cells at 24 hr Post-infection. In this experiment, male C57BL/6J mice (N = 3 - 4/group) were infected i.p. with 105 CFU of L. monocytogenes or were left un-infected. Mice were also administered the drug IS001 or vehicle (0.5% carboxymethyl cellulose) at the same time of inoculation and 12 hr later. Twenty-four hours after inoculation, mice were euthanized and the spleens were removed and were processed individually and stained for flow cytometry. Shown are the mean &#177; SEM frequency of IFN-&#947;+ cells in the NK (a) or NKT cell gates (b) in uninfected or infected mice after treatment with a vehicle or the drug IS001. *Indicates a difference (P &#60; 0.05) from vehicle control by two-tailed T-test. Data are re-printed from31 with permission from the Journal of Immunology (volume 195, pp. 5189-5202, 2015). Copyright 2015. The American Association of Immunologists, Inc. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54554/54554fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54554/54554fig5.jpg" /> 
Figure 5. Gating Strategy for Detection of IFN-&#947; Production in CD4 and CD8 cells. First gate on lymphocytes on FSC-A by SSC-A plots. Then gate on those events that are on the diagonal on the FSC-H/FSC-A plot. These are the singlets. Within this gate, gate on live (AmCyan-) CD45+ cells. Then gate on either CD8+ or CD4+ populations. Within each gate, the IFN-&#947;+ cells in the PE channel are identified by comparing the staining to the FMO control. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54554/54554fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54554/54554fig6.jpg" /> 
Figure 6. Representative Data Obtained for the Frequencies of IFN-&#947;+ CD4+ and CD8+ T Cells at 7 Days Post-infection with L. monocytogenes (EGD strain). In this experiment, male C57BL/6J mice were infected i.p. with 2 x 104 CFU L. monocytogenes (N= 7/group) or were left uninfected (N= 3/group). Mice were also administered the drug IS001 or vehicle (0.5% carboxymethyl cellulose) twice daily starting on the day of inoculation. Seven days later, mice were euthanized and the spleens were removed and were processed individually for cell culture. Splenocyte mononuclear cells were stimulated for 24 hr with heat-killed L. monocytogenes with protein transport inhibitor added for the final 4 hr of culture. Cells were then stained for flow cytometry. Shown are the mean &#177; SEM frequency of IFN-&#947;+ cells in the CD4+ (a) or CD8+ cell gates (b) in uninfected or infected mice after treatment with a vehicle (0.5% carboxymethyl cellulose) or the drug IS001. * indicates a difference (P &#60; 0.05) from the vehicle control counterpart as determined by two-tailed T test. Data are re-printed from31 with permission from the Journal of Immunology (volume 195, pp. 5189-5202, 2015).Copyright 2015. The American Association of Immunologists, Inc. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54554/54554fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54554/54554fig7.jpg" /> 
Figure 7. Representative Data Obtained during an Experiment that Compared the Effect of a PPAR&#945; Antagonist 1S001 on Mouse Survival to Endpoints after Infection of Male C57BL/6J Mice with L. monocytogenes (EGD strain). In this experiment, male C57BL/6J mice (N = 10 mice/group) were infected i.p. with the modified LD50 dose of the pathogen (105 CFU) of L. monocytogenes. Mice were also administered the drug IS001 or vehicle (0.5% carboxymethyl cellulose) twice daily starting on the day of inoculation. Mice were followed daily for clinical signs and were euthanized if humane endpoints were met. Shown is the percent survival of mice to endpoints over time * indicates a difference in the survival between groups as determined by log-rank test (P &#60; 0.05). Data are re-printed from31 with permission from the Journal of Immunology (volume 195, pp. 5189-5202, 2015). Copyright 2015. The American Association of Immunologists, Inc. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54554/54554fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/54554/54554table1.jpg" /> 
Table 1: Shows Some Representative Calculations for Determining CFU in an Aliquot of Day Culture. In this example, an aliquot of day culture was taken and was diluted 1:1 with BHI media. The OD600 of this diluted sample was determined to be 0.84. In addition, a 100 &#181;l aliquot was taken for CFU determination. This sample was diluted with 900 &#181;l of BHI media (10-1) and was washed and resuspended in 1 ml BHI. A 10-fold dilution series of this sample was prepared (10-2 to 10-9) and diluted samples were plated on BHI agar plates (only values for 10-4 to 10-9 are shown). The next day colonies were counted. Only those plates that had colony numbers between 30-300 were considered for the calculation (i.e., 10-6 plate, highlighted in yellow). The number of colonies on this plate (70) was then divided by 0.1 (volume in ml plated) to get the CFU/ml of the diluted sample. This value was then multiplied by the dilution factor (106) to obtain the CFU/ml reading of the undiluted culture. TMTC = too many to count.
<img alt="Table 2" src="/files/ftp_upload/54554/54554table2.jpg" /> 
Table 2: Staining Panel for Detection of IFN-&#947; in NK and NKT Cells. Note that either compensation beads stained with the flow antibodies used in the panel or splenocytes stained with various fluorochrome versions of CD4 antibody clone GK1.1 can be used as single positive controls.
<img alt="Table 3" src="/files/ftp_upload/54554/54554table3.jpg" /> 
Table 3: Staining Panel for Detection of IFN-&#947; in CD4+ and CD8+ Cells. Note that either compensation beads stained with the flow antibodies used in the panel or splenocytes stained with various fluorochrome versions of CD4 antibody clone GK1.1 can be used as single positive controls.

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 Listeria monocytogenes as a Model for Studying Host Interferon-&#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.

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 ...

experimental-infection-with-listeria-monocytogenes-as-model-for

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

13.5K Views.  University of Toronto.  Dit protocol beschrijft het enten van C57BL / 6J muizen met de EGD stam van Listeria monocytogenes (L. monocytogenes) en interferon-γ (IFN-γ) responsen door natuurlijke killer (NK) cellen, natuurlijke killercellen (NKT) cellen te meten, en adaptieve T-lymfocyten na de infectie. Dit protocol beschrijft ook hoe om te overleven onderzoek bij muizen na infectie met een aangepaste LD 50 dosis van de ziekteverwekker. 

Video: Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-γ Responses

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Introducing Shear Stress in the Study of Bacterial Adhesion

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

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

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

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Listeria monocytogenes Infection of the Brain

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. monocytogenes to cross the blood-brain barrier (BBB) is concerning as it can lead to life-threatening meningitis and encephalitis. The BBB protects the brain microenvironment from various toxic metabolites and microbial pathogens found in the blood following infection, and therefore supports brain homeostasis. The mechanisms by which L. monocytogenes present in the bloodstream cross the BBB to cause brain infections are not fully understood and there is also a lack of a robust model system to study brain infections by L. monocytogenes. Here, we present a simple mouse infection model to determine whether bacteria have crossed the BBB and to quantitate the burden of bacteria that have colonized the brain in vivo. In this method, animals were infected intravenously with L. monocytogenes and were humanely euthanized by exposure to CO2 followed by cervical dislocation. Cardiac perfusion of the animals was performed prior to harvesting infected organs. Blood was collected before perfusion and the number of bacteria per organ or mL of blood was determined by plating dilutions of the blood or organ homogenates on agar plates and counting the number of colonies formed. This method can be used to study novel receptor-ligand interactions that enhance infection of the brain by L. monocytogenes and can be easily adapted for the study of multiple bacterial pathogens.

Listeria monocytogenes Infection of the Brain

During infection, Listeria monocytogenes is capable of crossing the blood-brain barrier to colonize the brain. In this protocol, we demonstrate how to assess bacterial colonization of organs following infection of mice. A procedure to perform whole organ perfusion for specific determination of bacterial numbers in the brain parenchyma is provided.

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond&#160;only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...

Assay for the Identification of Interferon-Gamma Secretion in Murine Lymphoid Organs

Discard the fixative solution, and add 5 milliliters of PBS for 5 minutes at room temperature, under gentle agitation. Then, replace the PBS with 5 milliliters of 30% sucrose and incubate for 12 to 24 hours to maintain the tissue morphology. Now, the organ sinks at the bottom of the plate.
To freeze the sample, put dry ice in a large receptacle and place a smaller receptacle inside, containing around 50 milliliters of pure methanol and a few pieces of dry ice. Gently dry the spleen on a lint-free wipe. Drip a drop of OCT compound at the bottom of the base mold and place the spleen inside the mold. Be careful not to produce any bubbles.
Add approximately 1 milliliter of OCT over the spleen. With forceps, deposit the base mold on the surface of the methanol in the dry ice bath, making sure the methanol does not touch the OCT. The OCT will thicken and become white when frozen. Remember to freeze the tissue as rapidly as possible to minimize artifacts.
On a cryo-microtome, set to the temperature of minus 21 degrees Celsius. Section the tissue to desired thickness around 10 micrometers. Use a brush to collect sections onto glass microscope slides, and inspect visually. Allow the sections to come to room temperature.
Draw a circle with a liquid blocker, for example, a PAP pen, around the tissue section outside the OCT. Once the tissue has dried, rehydrate the sample by dripping 100 microliters of PBS on the tissue section for 5 minutes. Then, remove the PBS from the section by aspiration, and add 100 microliters of blocking solution per sample section. Incubate in a covered wet chamber for a minimum of one hour at room temperature.
To stain with primary antibodies, replace the blocking solution with the prepared primary antibody mix for each sample. Incubate for four hours at room temperature or overnight at 4 degrees Celsius in a covered wet chamber, before washing according to the manuscript.
Now, dilute the secondary antibodies of interest to an optimum concentration, usually 2.5 micrograms per milliliter in the blocking solution. Remove the final wash solution from the section, and add the prepared secondary antibody mix on top of the section, and incubate for one to four hours at room temperature in a covered, wet chamber.
After washing with wash buffer according to the manuscript, remove the wash solution and perform a final wash with PBS. Aspirate the phosphate-buffered saline and allow the remaining PBS to evaporate without the section completely drying out.
Draw a circle around the section on the back of the slide. Then, place a drop of the mounting medium on top of the sample, making sure the medium covers the whole section, and carefully place a cover glass on top. Let the samples polymerize overnight at room temperature in the dark.
In the morning, place the slide under an inverted spectral laser-scanning microscope. Adjust the objectives to 10x/NA 0.40, or 60x/NA 1.4 for analysis of cytokines sub-cellular localization.

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-γ Responses

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