Applicazione di nanoparticelle fluorescenti per studiare rimodellamento del sistema Endo-lisosomiale da batteri intracellulari

The overall goal of this procedure is to study the remodeling of the end lysosomal system by intracellular bacteria using fluorescent nanoparticles as tracers. This is accomplished by first seeding heela cells in an eight well chamber slide and incubating them overnight. The second step is to infect the cells with salmonella and tarica.

Next, a pulse chase labeling of infected host cells is performed by incubation with gold BSA Rumine nanoparticles. The final step is to observe alterations of the host cell endosomal system by confocal microscopy. Ultimately, quantification of the intracellular distribution of nanoparticles is performed using the MRS software package.

The main advantage of this technique over existing methods like immuno microscopic, is that n infected cells are analyzed With method, can provide insight into the intracellular lifestyle of terica. However, it can also be applied to other intracellular pathogens such as Gaia species, Ella Prolia, or mycobacterium tuberculosis. It may also be applied to other host cell types such as micro feh, cell lines, or primary cells.

The synthesis of 10 nanometer gold nano particles or NPS requires two solutions solution A and solution B.To prepare solution A, add two milliliters of 1%aqueous gold chloride into 160 milliliters of milli Q water. Prepare solution B by adding eight milliliters of 1%trisodium citrate dihydrate, and 160 microliters of 1%tannic acid into 32 milliliters of milli Q water. Warm up solutions A and B to 60 degrees Celsius and then mix them while stirring.

A dark blue color should be observed immediately after about 15 minutes. The solution should become red in color at this point. Heat the solution to 95 degrees Celsius.

Keep it at 95 degrees Celsius for five minutes, and then cool the solution to room temperature. Prepare the golden piece for coating and labeling by adding 900 microliters of gold MPS into a 1.5 milliliter einor tube and centrifuging at 15, 000 Gs For 30 minutes. Discard the supernatant and resuspend the pellet in 900 microliters of sterilized MQ water.

Add 100 microliters of A BSA solution and mix on a vortex at 800 RPM for 30 minutes. Remove excess BSA by centrifuging the preparation at 15, 000 Gs for 60 minutes at four degrees Celsius. Discard the supernatant and resuspend the gold BSA NPS and 125 microliters of PBS.

Add 12.5 microliters of one molar bicarbonate. The R domine n hydroxy CIN ester or NHS solution is prepared in DMSO immediately before. Use at 15 microliters of R DOMINE N-H-S-D-M-S-O solution to 0.5 milliliters of the gold BSA NP suspension.

Incubate the reaction for two hours at room temperature while mixing at 800 RPM and avoiding exposure to light. Next, purify the gold BSA rumine NPS through dialysis against PBS at four degrees Celsius with five buffer changes over a period of 36 to 48 hours after purification, stabilize the NPS by adding 10 microliters of B-S-A-P-B-S into each one milliliter of gold BSA RUMINE NPS to remove the free or released BSA Rumine centrifuge at 15, 000 GS for 16 minutes at four degrees Celsius. Resus suspend the pellet in two milligrams per milliliter of B-S-A-P-B-S after measuring the optical density at 520 nanometers or OD five 20.

Store the gold BSA Rod Domine NPS at four degrees Celsius, avoiding exposure to light the he a cells for this experiment permanently express a green fluorescent protein tagged lysosomal glycoprotein lamp one GFP and are cultured in DMEM with 10%fetal calf serum at 37 degrees Celsius in an atmosphere containing 5%carbon dioxide seed the cells at a density of 50, 000 per well in an eight well chamber slide and incubate overnight. The bacterium for infecting the hela cells is the human gastrointestinal pathogen, salmonella and tarica. For comparison, two mutant strains are used.

All three strains harbor a plasmid for constitutive expression of enhanced GFP and are cultured in LB broth with the appropriate antibiotics to maintain the plasmids for each strain. Inoculate a single colony of bacteria in three milliliters of LB broth with the appropriate antibiotic and grow overnight at 37 degrees Celsius. Under shaking conditions for aeration on the following day, dilute each culture one to 31 in fresh LB broth and continue growing for three and a half hours.

To begin this procedure, measure the OD 600 of the subculture bacteria and dilute to an OD 600 of 0.2. In one milliliter of PBS, add appropriate amounts of bacteria to the hela cells in the A 12 chamber slide to achieve a multiplicity of infection of 100. Incubate for 30 minutes in the cell incubator.

Wash the hela cells three times with PBS to remove non internalized bacteria. This time point is set as zero hour post-infection or zero hour pi. Add 300 microliters of fresh culture medium containing 100 micrograms per milliliter of gentamicin to the cells and maintain for one hour.

After one hour. Replace the medium with imaging medium containing 10 micrograms per milliliter of gentamicin. Add gold BSA rumine NPS to the hela cells to obtain a final concentration of OD five 20 of 0.1.

Incubate for one hour after one hour. Remove the medium and wash three times with PBS. Finally, add 300 microliters of fresh imaging medium containing 10%FCS and 10 micrograms per milliliter of gentamicin.

For the rest of the incubation time, high resolution imaging of the cells at different time points is accomplished using a confocal imaging system such as a confocal laser scanning or spinning disc microscope with a humidified environment chamber switch on the temperature control system and wait until it is stable. Optimize the imaging settings such as the magnification scanning, speed resolution, and Z step size. Use appropriate excitation emission settings for GFP and gold BSA rumine NPS at indicated time points post-infection.

Mount the A 12 chamber slide containing infected cells on the microscope stage and record images to analyze the images and study the remodeling of the endosomal system by intracellular salmonella. Use image analysis software. Open the data by clicking open and choosing the file in the objects toolbar of the surpass view.

Click on the icon to add a new surface item. To analyze a region of interest or ROI select segment only a region of interest, then click next as a source channel. Select channel three.

Check the smooth option to set up the smoothness of the resulting area. Define a value manually or accept the automatically generated value. For threshold.

Select the absolute intensity option for the threshold adjustment. Select the manual option and set a value in the viewing area. A surface threshold preview is displayed in gray.

Click next on the tab Classifies surfaces. The resulting surface can be filtered by various criteria. A default filter is number of V holes greater than 10 other filters can also be included by clicking add.

In this example, the default filter is used to complete the surface creation. Click on finish in the object list. Uncheck the box for the item volume.

A new created surface is now displayed in the viewing area. In red, export the statistics to an Excel file by clicking save. The gold NPS synthesized by this protocol were quasi spherical in shape and approximately 10 nanometers in size.

BSA coating and rho domine labeling did not influence their morphology or size. To study remodeling of the host cellular endosomal system by intracellular salmonella. Hela cells were mock infected or infected with wild type salmonella or two mutant strains and pulse chased with 10 nanometer gold.

BSA rod domine n peas in non-infected cells, the NPS uniformly distributed in late endosomes or lysosomes. In contrast, the NPS in wild type salmonella infected cells were largely rearranged at eight hours post-infection. A stabilized network of salmonella induced filaments or sifts was formed, and most mps were found accumulated within the tubular structures.

The mutant strains exhibited distinct behaviors at eight hours post-infection. The SSAV mutant strain was confined inside the salmonella containing VAE or SCV. While no sifts were formed, the majority of mps were still located in free late endosomes or lysosomes for the sif.

A mutant strain escape of salmonella into the cytoplasm occurred and no association between NPS and bacteria was observed. When accessibility of the end lysosomal system at various phases of salmonella infection was examined, the results show that in all cases, most of the internalized NPS accumulated in the SCV or SIFs Following this procedure. Other methods, neck transmission microscopic can be performed.

This may answer additional questions like ultra structure or membrane compartments in some nano infected host cells. After watching this video, you should have a good understanding of how to prepare fluorescent nanoparticles. These can be used to label hoster compartments modified by intracellular salmonella.