The overall goal of the following experiment is to assess the level of colocalization of internalized bacteria, RIA Meningitidis with the cytoskeletal protein Alpha Actinin. This is achieved by infecting cultured human brain microvascular cells with an overnight culture of bacteria for three to eight hours to allow the bacteria to enter the target cells. As a second step cells with intracellular bacteria are washed, fixed, and then permeated, which prepare the infected culture for immuno staining of internalized bacteria and intracellular alpha actinin.
Following the confocal imaging and application of colocalization software, images of the intracellular colocalization can be observed and quantified results are obtained that show Meninga Croci expressing OPC and also membrane protein can specifically interact with intracellular alpha actinin based on confocal microscopic visualization and by quantitative colocalization analysis. Hi, I'm Isel Maria from Professor Mata TI Lab at the Department of Cellular Molecular Medicine at the University of Bri. Today we will show you a procedure for estimation of the level of col, ratio of intracellular bacteria with cytoskeletal proteins.
We use this procedure in our laboratory to study an interaction of mycelium meningitis expressing an auto membrane protein OPC with the cytoskeletal protein alpha actin. So let's get us started. This work needs to be done in an adequate safety laboratory.
Two days prior to the immuno staining procedure, inoculate the bacterial stain of interest on brain heart infusion. Agar plates supplemented with 10%heated horse blood grow overnight at 37 degrees Celsius in a 5%carbon dioxide atmosphere on the day before immuno staining. Use a 10 microliter culture loop to make a suspension of the overnight bacterial culture.
In two milliliters of PBSB, leave the suspension to stand for five minutes at room temperature to allow large bacterial aggregates to settle. Next, transfer the top one milliliter of the suspension into a sterile tube without disturbing the pellet solubilize bacteria By adding one milliliter of 1%SDS 0.1 molar sodium hydroxide to two vials containing 20 microliters of bacterial suspension and invert the tube to mix. To estimate bacterial numbers.
Measure the nucleic acid content by determining the absorbance of the solution. At 216 anters adjust the bacterial suspension to the required density in infection medium for infecting the human brain microvascular endothelial cells. Human brain microvascular endothelial cells or H-B-M-E-C-A seeded two days prior to immuno staining placed 16 millimeter glass cover slips into the wells of a 12 well plate and seed HBME seas at 50%Confluence on the cover slips incubate overnight at 37 degrees Celsius in 5%carbon dioxide atmosphere on the following day.
Infect cells with the freshly prepared bacterial suspension in our laboratory. An infection ratio of 200 to 300 colony forming units per target cell is routinely used. Incubate for three to eight hours at 37 degrees Celsius in 5%carbon dioxide at the end of the infection period.
Wash cells three times with PBS and fixing 500 microliters of 2%para formaldehyde for 30 to 45 minutes at room temperature After washing off the para formaldehyde, perme the paraldehyde fixed cells by incubating in 0.1%Triton X 100 diluted in PBS for 10 minutes. Finally wash the samples three times with PBS and proceed to the immuno staining as shown. Next staining of intracellular bacteria and dfor actinin can be performed simultaneously.
In 12 well plates block the cover slips containing the permeable cells with 500 microliters of 3%B-S-A-P-B-S-T for 30 to 60 minutes. At room temperature after washing with PBS transfer, each cover slip to a new dry well in the 12 well plate at the primary antibodies against bacteria and alpha actinin simultaneously 80 to 100 microliters of antibody per cover slip is sufficient if added carefully to cover the surface of the cover slip, incubate for one hour at room temperature At the end of the incubation, add 200 microliters of PBS to the, well lift the cover slip and place it in a new well containing 500 microliters of PBS After five minutes, remove the PBS by pipetting and then add fresh PBS. Repeat twice and transfer cover slips to a new dry well.
Next, add the appropriate secondary antibodies conjugated to different fluorochromes diluted in 1%B-S-A-P-B-S-T Incubate room temperature for one hour in the dark at the end of the incubation wash is shown previously then counterstain DNA with DPI for five minutes at room temperature in the dark at the end of this incubation wash the cover slips in PBS once and then mount with a drop of mounting. Medium mounted cover slips should be stored in the dark until they're ready for observation. Under the microscope, we observe and capture images of our immuno labeled samples using a confocal laser scanning microscope attached to an inverted epi fluorescence microscope.
To begin the CLSM procedure at a drop of immersal to the objective and place the specimen slide cover, slip down on the microscope stage, set the microscope to a visual mode and find the area of interest using the eye pieces of the microscope, we are now ready to use the Leica software. Select the XY zed acquisition mode. Select the five 12 by five 12 format for colocalization studies.
The higher the resolution, the more accurate the image. Then select the bidirectional X mode, which will increase the scanning speed and help reduce photo bleaching. Set up sequential scanning settings.
Click in the SEC function and then select between lines. Select laser beams according to the fluorochromes conjugated to the secondary antibodies. 4 0 5 nanometers for DPI 4 88 nanometers For Alexa, Fluor 4 88 and 5 61 nanometers.
For trixy activate photomultiplier tube one, two, and three respectively. Set up top and bottom of Z stack or series. Next set, Zed step size to 0.2 micrometers.
RIA Mencius is a diplo occus and since each caucus has an approximate diameter of 0.5 micrometers, a Z step size of 0.2 micrometers enhances the probability of scanning each caucus at least twice. Set the final scan parameters by selecting line averaging of three to improve the signal to noise ratio by clicking start. Double or triple stained images are obtained by sequential scanning at different wavelengths and by using the between lines mode for each channel to eliminate crosstalk between the different chromophores.
For an indication of colocalization of two fluorochromes, select the overlay function to merge selected channels into a single image. For example, when both Alexa 4 88 and trissy Fluor Chromes co localize yellow color will appear in the overlaid image for A 2D reconstruction required for visualizing possible colocalization compiles Zed stack or series using the maximum projection function. After acquiring the Z stack or series process your data to obtain an orthogonal image for visualization of intracellular localization of various elements.
Quantitation of colocalization is performed using Vol City software from IMP provision. To begin create a library with CLSM images, select extended focus from the image in the top bar. This tool will combine Zacks in a 2D image to be analyzed.
Now select the COLOCALIZATION tool. The two channels to be analyzed should have the same color depth. Select the area that is going to be quantified.
Set the threshold to remove any background. Create colocalization output by selecting generate colocalization. Colocalization Statistics will be generated for the regions of interest selected previously.
Next, select mander's coefficients R and ny. Finally, export or statistics values to an Excel document for data presentation shown here a representative confocal imaging results of human brain endothelial cells infected with meninga cockeye for eight hours. The location of bacteria is shown in red.
While the location of alpha actinin is shown in green arrows indicate regions where a high degree of alpha actinin accumulation appears to have occurred around bacteria. In this overlay image, there are several regions in which yellow orange color appears suggesting colocalization between meninga occi and alpha actinin. In this next image, optical dissection of an infected H-B-M-E-C monolayer indicates colocalization around intracellular bacteria located at the base of a cell.
When three dimensional images of infected H-B-M-E-C Monolayers were processed, an oblique view of the apical surface shows a Durant bacteria stained red indicated by the red arrow where several bacteria located towards the basal surfaces of endothelial cells indicated by the yellow arrow. A distinctly orange, yellow in color basal location can be more clearly seen in this endon XZ cross-section. In contrast to afar, actinin experiments in which labeling of internalized bacteria and either menton or actin was performed reveal occasional colocalization with actin but rare colocalization with fermenting in H-B-M-E-C cells.
As observed in the previous experiments, Retin was concentrated around several internalized bacteria indicated by the white arrows. The data were also analyzed to obtain relative overlap. Coefficient R, which according to Manda, represents the true degree of colocalization IE, the number of pixels that co localize compared with the total number of pixels and NY values for alpha actinin, menting and actin overall in H-B-M-E-C infected with OPC expressing meningococci, greater than 25%overlap of rein in a meningococci was obtained.
The coefficient and Y is a measure of the frequency of a currency of the more abundant rein signal each time the less abundant meninga cocoa signal occurs. This measure shows a striking level of occurrence of alpha actinin in the vicinity of internalized meninga cockeye. We just show you how to visualize and quantify introduction of internalized bacteria with cytoskeleton elements within this experiment.
It's important to remember to follow the fixing and immune stem protocols strictly to maintain the integrity of the structures and to avoid a high background. So that's it. Thank you for watching and good luck with your experiments.
