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PBS injected through di peso sana portal vein will exit from the severed inferior vena cava. Liver perfusion ensures that harvested cells are from the liver and not the circulating blood.
Remove the perfused liver from the mouse's body cavity, place in FACS buffer, and store on ice. Go to step 5. Remove the spleen from the mouse, place in FACS buffer, and store on ice. Weigh the whole spleen, cut in half, and weigh one of the halves.
Place one half in FACS buffer and go to step 6. Place the other half in a Stomacher bag on ice and go to step 7. When perfused properly, the liver turns from a dark red color to light brown after injecting please click for source PBS.
If the liver is not required for FACS analysis, perfusion is not necessary. The liver can be isolated and collected directly into a Stomacher bag and processed as described in step 7. For euthanasia of mice, CO 2 asphyxiation is the preferred method because it has minimal impact on bacterial CFU and immune cell viability in the spleen and liver.
If blood collection is not required, cervical dislocation may be used as alternative euthanasia method. It is recommended dr dale gray perdita di peso euthanasia methods that may affect organ weight or immune cell viability not be used eg thiobarbiturates. Push the tissue through the cell strainer using the plunger of a 5 mL syringe until a single-cell suspension is formed.
Transfer the hepatic single-cell suspension from the Petri dish to a 50 mL conical screw-cap tube and dr dale gray perdita di peso the total volume to 40 mL with cold FACS buffer. Vortex cell suspension and take a 1 mL aliquot to determine bacterial load for the liver. Go to step 7. Resuspend the cell pellet in 20 mL isotonic Percoll at room temperature. Discard supernatant and disc-like sheet of cells floating on top i.
Dilute cell suspension in trypan blue depending on suspension volume in step 5. Liver single-cell suspensions can then be labeled with antibodies specific for desired cell-surface markers and analyzed by FACS. Preparation of splenic single-cell suspension for FACS analysis Generate single-cell suspensions by placing one half of the spleen step 4.
Transfer the spleen single-cell suspension from the Petri dish to a 10 mL centrifuge tube and bring the total volume to 10 mL using cold FACS buffer. Dilute cell suspension - in trypan blue 0. Count viable splenocytes using hemacytometer - see Step 5. Measurement of bacterial load in tissues from Listeria -infected mice Fold the top of the Dr dale gray perdita di peso bag containing the spleen half.
While holding the Stomacher bag shut to prevent leakage, lay it flat on a clean bench or dr dale gray perdita di peso a laminar flow cabinet.
Roll a thick round pen over the tissue until the tissue is mashed within the bag. Add 5 ml of PBS to each Stomacher bag containing mashed tissue. Mix the homogenate in the Stomacher bag or the 1 mL aliquot of hepatic cell suspension with a pipette.
Perform the following in duplicates. Carefully spread 0. Count the number of CFU for each dilution and calculate the CFU per tissue taking into account the portion of the tissue e. Tips dr dale gray perdita di peso notes: More than one Stomacher bag can be placed in the Stomacher at a time as long as total volume dr dale gray perdita di peso not exceed maximum volume as specified by the manufacturer If a Stomacher is not available, a tissue homogenizer can be used instead.
Alternatively, the following method, although not as efficient, can also be used to manually macerate the organ in place of steps 7. Roll a mL laboratory glass bottle repetitively over the bag until the tissue is thoroughly mashed.
Add 5 mL of PBS to each bag and proceed to step 7. Ma deve essere un medico a fare una ricetta precisa che riporti il dosaggio desiderato di tiroide secca o ormoni, che poi il farmacista prepara. Several studies suggest that about one-fifth of with POI have an autoimmune disease. Find a Location. Microsoft PowerPoint - Work together on. Non riesci ancora a vedere i tuoi contatti o il tuo credito Skype Prova ad accedere con un qualsiasi indirizzo e-mail che potresti avere usato in precedenza per accedere a uno qualsiasi dei servizi Microsoft.
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An unexpected error occurred. Issue 54 doi: dr dale gray perdita di peso 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. Wang, N. Listeria monocytogenes Listeria is a Gram-positive facultative dr dale gray perdita di peso pathogen 1. Mouse studies typically employ intravenous injection of Listeriawhich results in systemic infection 2.
Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells 5.
During the dr dale gray perdita di peso three days of infection, different go here immune dr dale gray perdita di peso e. Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses 6. There is a broad range of sensitivities amongst inbred mouse strains 7,8. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection 6, Determination and comparison of more info 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.
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 Listeriaand the relevant institutional biosafety committee and animal facility management should be consulted before work commences. Culturing and long-term storage of Listeria monocytogenes Listeria. Preparation and storage of Listeria infectious stock for in vivo infection studies. Preparation of Listeria inoculum and intravenous injection of mice.
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Removal of liver and spleen from Listeria -infected mice for analysis. Measurement of bacterial load in tissues from Listeria -infected mice. For a standard infection experiment, Listeria was obtained from a frozen glycerol stock and streaked on a HBA plate as shown in Figure dr dale gray perdita di peso.
If few colonies are obtained after streaking, this may indicate poor streaking or a less than optimal frozen stock. We observe that mice infected with sub-lethal doses of Listeria using this protocol may transiently exhibit ruffled fur, hunched posture and weight loss within the first few days. These symptoms provide a visual cue as to how severely an individual mouse is affected by the infection, whether it responds differently to the rest of the group i.
In the results represented by Figuresmice were infected using a freshly thawed aliquot of Listeria infectious stock. At different time points post-infection, mice were euthanased and liver and spleen were removed. Figure 2 shows a HBA plate on which diluted spleen homogenate from a single mouse was cultured. There should be a clear reduction in the number of colonies as the dilution factor becomes higher, such that counting distinct CFU is possible for at least two dilutions.
If the mouse has cleared dr dale gray perdita di peso Listeria infection i. Figure 4 shows cell counts based on single-cell suspensions prepared from spleen and liver of infected mice at different time dr dale gray perdita di peso after infection. Leukocyte purity can be determined by FACS analysis using a monoclonal antibody specific for the pan-leukocyte marker CD45 clone F Typically, the number of splenocytes and hepatic leukocytes increase during the period it takes to clear the infection with the liver exhibiting a greater fold increase in hepatic leukocytes, but a substantially smaller total, compared to the increase of splenocytes in the spleen.
The type go here number of the immune cells can be determined by labeling the single-cell suspensions with antibodies specific for cell-surface markers.
Labeled cells can then be detected by FACS analysis. Figure 1. HBA plate dr dale gray perdita di peso with Listeria. A sterile inoculating loop was used to streak Listeria from a frozen glycerol stock. Figure 2. Tissue homogenate from a Listeria dr dale gray perdita di peso mouse cultured on HBA plates. The dilutions that enable counting of individual colonies are used to determine the bacterial load i. Figure 3. At each time point, mice were euthanased, the liver was perfused and harvested with the spleen, and dilutions of splenic homogenate A or hepatic single-cell suspension B were cultured on HBA plates to determine the bacterial load.
Solid lines indicate geometric mean, and vertical bars indicate SEM. Figure 4. Viable cell counts for tissues from infected mice.
At each time point, mice were euthanased, the liver perfused and harvested with the spleen.
Cells were stained with trypan blue and counted using a hemacytometer as described in Step 5. Splenocyte B and hepatic leukocyte Dr dale gray perdita di peso counts obtained from single-cell suspensions prepared from Listeria -infected mice. Lines indicate geometric mean. Figure 5. Listeria is one of the most widely used organisms to characterize host immune responses to intracellular bacteria 6. The protocol presented here enables one to measure bacterial load and immune cell responses within the same tissue of a given mouse.
This dual measurement of a particular tissue for each infected mouse provides for more robust comparisons within and between mouse cohorts either representing different mouse strains or time points post-infection. While Listeria infection by oral administration could also be used to study immune responses in mice, infection by intravenous injection is often used because: 1 it ensures rapid and effective delivery to the bloodstream; 2 it results in a synchronized and consistent systemic infection; and 3 dr dale gray perdita di peso Mus species harbors a mutation in the gene encoding the E-cadherin receptor, which limits Listeria infection by oral administration this mutation affects Listeria 's ability to bind the mouse E-cadherin receptor and to efficiently cross the epithelial lining of dr dale gray perdita di peso gastrointestinal tract There are a few critical steps in this protocol.
First, the Listeria inoculum stock should be generated from a fresh overnight HBA culture to ensure viability and virulence. Second, it is important to accurately determine the CFU concentration of the inoculum before and after injecting all mice to ensure that the CFU concentration does not differ greatly dr dale gray perdita di peso the first and last mouse injected.
Third, injection of Listeria into the tail vein must be consistent for all mice. Lastly, it is necessary to perfuse the liver to deplete non-resident leukocytes to ensure accurate measurement of immune cells within the liver and not leukocytes passing through in the peripheral blood.
Two limitations of this protocol are the investigator's skill for infecting mice by intravenous injection and the detection of bacterial load in tissues.
If one person is injecting a large number of mice, Listeria viability i. CFU concentration may reduce over time once the frozen inoculum is thawed e. If a Stomacher is not available, then alternative methods to homogenate the tissue, such as using a tissue homogenizer, can be used instead for steps 7.
However, room temperature should not be used when growing cultures for preparation of frozen Listeria stocks. This protocol provides a basic approach for characterizing Listeria infection in mice and can be used with other Https://durante.onlinecasinoslots.icu/num5734-fat-burn-tapis-roulant.php strains besides EGD. In addition to the liver and spleen, single-cell suspensions from lymph nodes can also be generated.
In either instance, these single-cell suspensions can be used for a variety of analyses, including measuring immune cell subsets, and in vitro stimulation of sorted dr dale gray perdita di peso cells. Once the basic techniques are mastered this protocol can also be modified to isolate Listeria -specific T cells 19characterize dendritic cells 20or perform immune cell depletion at certain time-points after infection 21,22 to more thoroughly characterize the immune response in mice infected with Listeria.
NW is supported by an Australian Postgraduate Award. OW is supported by a R. You must be signed in to post a comment. Please sign dr dale gray perdita di peso or create an account. This content is Open Access.
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Gmail and other free mail providers are ineligible for trials. Please, sign in with Google or fill out the form below to receive a free trial. Dr dale gray perdita di peso and Infection. Culturing and long-term storage of Listeria monocytogenes Listeria Make or purchase pre-made horse blood agar HBA plates.
Obtain viable Listeria from one of the following: an infected mouse tissue homogenate, a lyophilized stock, a frozen glycerol stock, or a colony from a recent Listeria culture i. Link Listeria should be obtained using a sterile inoculating loop and employing aseptic techniques for all methods described in this protocol.
Make a series of unidirectional streaks from the primary spread. Repeat streaking times using a fresh or re-sterilized loop before each set of streaks. Pick a single colony from a fresh Listeria culture on a HBA plate using a sterilized inoculating loop and inoculate 10 mL of brain heart infusion BHI broth prepare according to manufacturer's instructions in a 30 mL McCartney bottle.
Transfer 0. Contaminating growth can be minimized in culture by adding antibiotics to the media for Listeria strains with defined antibiotic resistance e. If Listeria is obtained from an untested source, it is recommended that additional tests be performed to confirm the dr dale gray perdita di peso of Listeria L.