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The overall goal of this procedure is to investigate intracellular mechanisms that control bacterial dissemination during infection in vitro and in vivo by using tissue culture cells in zebra fish larvae. The in vitro method begins with preparation of cha flexner, a gram-negative and enteroinvasive Bacterial pathogen Heller are then infected and irna or pharmacological reagents are used to manipulate the host autophagy machinery and cytoskeleton during infection. To study autophagy cytoskeleton interactions caused by cha in vivo zebrafish embryos are infected three days post fertilization for either method.

The final step is to label tissues with autophagy and cytoskeletal markers. Ultimately, results will provide insights into the mechanisms required for the control of infection by cytosolic host responses. The advantage of using gel to study bacterial autophagy over other bacteria such as listeria, is that gel targeted autophagy can also be entrapped incepting cages.

This method can help answer key questions in the autophagy field, such as what is the role of the cytoskeleton in autophagy and what is the role of autophagy in the restriction or promotion of intracellular bacteria? The implications of this technique can extend towards other processes of autophagy, such as the autophagy of viruses or mitochondria, and can also have therapeutic implications for human diseases with a septin or autophagy component. These are demonstration of zebra fish.

Infection by shige is critical as the steps involving injection of zebra fish with bacteria are difficult to learn because needle preparation and proper injection takes time and high resolution. Microscopy of zebra fish infection takes experience For this protocol. Use the wild type strain of cha Flexner M nine DT from a frozen glycerol stock streak bacteria onto Congo Red triptych soy plates, and incubate them at 37 degrees Celsius overnight.

Also, seed hella cells in six well plates in MEM media with 10%fetal calf serum the following day. Pick an individual colony and grow it up in TCS media overnight with shaking at 37 degrees Celsius the next day. Set up subcultures by making one to 80 dilution in fresh TCS.

When the optical density of the subcultures at 600 nanometers is between 0.3 and 0.6. Collect the bacteria by spinning them down at 1000 G for five minutes. Then reconstitute the bacterial pellet in MEM to the same optical density.

Hello, cells should be infected with a multiplicity of infection of 100 to one directly. Add the chag diluted in MEM to the plated hella cells and bring the total MEM volume to two milliliters per well. Then to maximize bacterial adherence to the host cells, centrifuge the plates at 700 G for 10 minutes and at room temperature, incubate the plates for 30 minutes so the bacteria can infect the cells after the infection period.

Gently wash the cells twice. Using fresh MEM to further eliminate extracellular bacteria, incubate the cells in Gentamycin containing complete media for one to four hours based on empirical observations. Next, fix and label the infected cells.

Image them by epi fluorescent or confocal microscopy using a 63 x or 100 x objective to resolve the DAPI labeled cha septin cages can be visualized as ring-like structures about 0.6 microns and diameter surrounding cytosolic bacteria polymerizing acton. The bacteria inside the septin cages can also be recognized by autophagy markers like P 62 and lc three a TG eight. Conceptually, the most difficult aspect of this procedure is the identification of bacterial autophagosome and sing cages in vivo and in vitro, which requires careful microscopy and analysis.

Moreover, one must ensure that the proper number of events over several biological replicates are being analyzed and ensure statistical significance After infecting the hella cells with chila. First, allow the bacteria at least two and a half hours to find their way to the cytosol. Next, the drug treatment can be made to treat the cytoskeleton.

Add between one to five microliters of diluted drug stock to each two milliliters of media with cells as a negative control. Treat one set of cells with DMSO in MEM, then incubate the cells for 30 minutes to manipulate the autophagic flux of the cells. Use 20 nano molar rapamycin or 160 nanomolar bafi mycin.

Then incubate the cells for four to 12 hours after the treatment. Fix and label the cells as described in the text protocol. This technique models gel infection and autophagy cytoskeleton interactions in vivo To begin subculture and collect gel as previously described, wash the bacteria with one XPBS and centrifuge them at 1000 G for 10 minutes.

Then resuspend the bacteria in 80 microliters of 0.1%Phenol red in PBS Keep this preparation on ice. It should have about 2000 bacteria per nanoliter 72 hours before the planned injection. Collect embryos from spawning zebrafish to remove any extracellular contaminants.

Wash the embryos in E two media with 0.003%bleach for 10 minutes. After 10 minutes, change to E two media and wash the embryos five times in E two. Next load 10 centimeter dishes with 50 milliliters of E two and up to 50 embryos.

Grow up a total of about 100 embryos at 28 degrees Celsius, 24 hours post fertilization. Treat the developing embryos with 0.003%and pheno threa in E two to prevent mein, which interferes with microscopy. Begin with anesthetizing the 72 hour larvae using a 200 microgram per milliliter trica bath in E two media.

Then using a micro manipulator and fine tweezers, break the tip of the micro injection needle and check the brake under a microscope. Now load the needle first, transfer a drop of the bacterial culture to a cover slip on the microscope stage. Second, turn on the micro injector and gas cylinder.

Third, lower the needle tip into the drop. And fourth, fill the needle to assess zebrafish survival during cha infection. Use a coddle intravenous injection using a fine paintbrush.

Position the anesthetized larvae laterally on the injection plate with their dorsal sides facing the needle. Now the needle tip must be positioned close to the urogenital opening aimed at the coddle vein. Then pierce the skin and inject a one in five nanoliter bolus of bacteria.

A successful injection into the codal vein is indicated by visualizing phenol red extending along the vein. Technically, the codal vein infection is the most difficult aspect of this procedure, and we require practice zebrafish larva. Confocal microscopy can also be challenging because the larvae require proper ing.

After the injections transfer each infected larvae individually to a well of a 24 well plate containing one milliliter of E two media. Incubate the plate at 28 degrees Celsius and monitor the embryos daily. To visualize recruitment of septin and autophagy markers.

Inject Ella at localized sites such as the tail muscle. Prepare to inject the larvae as shown in the previous section. Orient the zebra fish laterally and place the needle over the muscle.

Somites then pierce the space between two somites and inject about one nanoliter of Ella. A successful injection into the tail muscles will show that the injected so mite colors in red. As a consequence of the phenol red buffer, have prepared low melting 0.1%agarose in E two media at 37 degrees Celsius.

Add drops of agarro to a glass bottom dish individually. Transfer the injected larvae with a minimal volume of solution to the drops of agarro. Orient them there as desired and let the agarro solidify.

Next, cover the whole dish with LMA followed by E two with trica. Then take Zacks every two minutes for several hours. In infected tissue culture cells, shaila escapes from the phagosome to the cytosol where the host cytoskeleton can rearrange and compartmentalize bacteria inside septin cage like structures.

Bacteria and trapped in septin cages are recognized by autophagy markers such as P 62 and lc three. The zebrafish represents a valuable new host for the in vivo study of CHA infection. Depending on the infectious dose, zebrafish can either clear or low dose within 48 hours post-infection, or in the case of a high dose succumb to infection.

The optical transparency of zebra fist RV enables in vivo visualization of septin cages and achievement that has not been performed using other vertebrate models to complement evidence that septin cages and trap bacteria targeted to autophagy. GFP lc three transgenic zebrafish were infected to observe the recruitment of lc three positive autophagosome to intracellular cha in vivo in real time to characterize autophagy P 62 morpho treated larvae were injected with chala control. Morpho larvae showed normal septin cage structures.

However, in the P 62 morpho treated larvae, septin recruitment to chala was reduced, indicating that P 62 is an essential component to the autophagy mechanism, him Once mastered the infection, PHI larva can be done in one to two when it is performed properly Following this procedure. Other methods such as genome editing using the crisp perca system can be used to manipulate zebrafish genome. In addition, generation of novel fluorescently target zebrafish line can be engineered to follow autophagy cytoskeleton dynamics in real time during gel infection using live imaging After its discovery.

The Chaga Septin cage has been used to study autophagy cytoskeleton interactions and has paved the way for researchers in infection and cell biology to illuminate new roles for the cytoskeleton in autophagy. Don't forget that working with Ella can be extremely hazardous and precautions such as working in a level two lab environment should always be taking while performing this procedure.