The overall goal of this high-throughput microscopy-based assay is to identify host factors involved in Brucella infection of HeLa cells. This method can help answer key questions in the host pathogen interaction field, such as identifying host proteins that either promote or prevent colonization by the intracellular pathogen Brucella. Main advantage of this technique is that the semi-automated workflow from the vet lab to image analysis allows for robots and efficient high-throughputs creating experiments.

So this method can provide insight into Brucella host interactions using siRNA screens. Most of the protocol can be applied to other screening strategies. For example, or siRNA libraries.

After culturing HeLa cells and preparing screening plates with siRNAs according to the text protocol thaw a black 384 well plate by centrifugation at 300 times G and room temperature for 20 minutes. Prepare transfection medium by using room temperature dmem without FCS. To dilute the transfection reagent one to 200 and carefully mix before use.

Using a reagent dispenser add 25 microliters of transfection medium to each well of the 384 well plate and mix the solutions by moving the plate back and forth. Incubate the plate at room temperature for one hour to allow siRNA transfection reagent complex formation. In the meantime prepare HeLa cells by aspirating the culture medium and adding 2.5 milliliters of 0.05%trypsin EDTA in PBS to wash the subconfluent cells of a 75 square centimeter flask once.

Then add 1.5 milliliters of fresh trypsin and transfer the flask to 37 degrees Celsius for two to three minutes until the cells round up. Use 10 milliliters of prewarmed dmem 60%FCS to resuspend the cells. Then, use an automated cell counter to count the cells before preparing a cell suspension of 10, 000 cells per milliliter in dmem with 16%FCS.

Add 50 microliters of cell suspension to each well of transfection reagent. Resulting in 500 cells per well. Move the plate back and forth to evenly distribute the cells.

Then, leave the plate at room temperature for five to 10 minutes to allow the cells to settle down. Use parafilm to seal the plate. And incubate it in a humid incubator on a prewarmed aluminum plate at 37 degrees Celsius with 5%C02 for 72 hours.

Working with the class three pathogen B.abortus is dangerous and local biosafety regulations need to be followed which includes wearing a protective overall suit, protective gloves, mask, and goggles. One day, prior to infection, inoculate the previously determined amount of B.abortus starter culture in 50 milliliters of TSB KM medium. In a 250 milliliter screwcap bottle.

Use parafilm to seal the bottle and grow bacteria overnight at 37 degrees Celsius and 100 RPM on a shaker to optical density 600.8 to 1.1. Measure the optical density 600 of the bacterial culture. And prepare the infection medium by diluting the bacterial culture in dmem 10%FCS to achieve the desired multiplicity of infection.

Or, MOI. Using an automated plate washer exchange the transfection medium with 50 microliters of infection medium. Inside the hood transfer the plate into the centrifuge cage and centrifuge the 384 well plate at 400 times G and four degrees Celsius for 20 minutes.

Then, use parafilm to seal the plate and incubate it on a prewarmed aluminum plate at 37 degrees Celsius and 5%C02 for four hours. After the incubation with the automated plate washer use dmem with 10%FCS containing 100 micrograms per milliliter of dentrimysen or GM to wash the cells to inactivate extracellular bacteria. Then, use parafilm to seal the plate and place it back on the prewarmed aluminum plate in the incubator for another 40 hours for the end point assay.

To fix the cells, first use PBS to wash the wells. Then, exchange the PBS with 50 microliters of 3.7%PFA. In 2 at pH 7.4.

And incubate the cells at room temperature for 20 minutes. After the incubation use 50 microliters of PBS to exchange the fixation medium. To stain the samples with the automated plate washer begin by using 50 microliters of 1%Triton X-100 in PBS for 10 minutes to permeabilize the cells.

After using PBS to wash the cells three times add 50 microliters of PBS with one microgram per milliliter of DAPI and incubate at room temperature for 30 minutes. Then, use PBS to wash the cells three times before protecting them from light. Transfer the plate to the automated microscope.

Set up the microscope parameters. Select the 10-X subjective. Then select the plate format corresponding to the 384 well plate.

Acquire nine sites per well. Select Enable laser-based focusing. And, focus on plate and well bottom.

Then, set first well acquired as the initial well for finding the sample and all sites for site autofocus. Next, for the DAPI channel manually adjust the Z offset for the focus. Then, press Find Sample to set the focus on the well bottom.

Using the AutoExposure function get a good estimate for the exposure time. Manually correct the exposure time for the DAPI channel to ensure a wide dynamic range with low overexposure. Use this exposure time to image several sites throughout the plate.

And adjust the exposure time manually until the brightest pixels reach approximately 80%of the maximal brightness over the sites. Repeat the steps to set the focus and exposure time, accordingly for all other channels. Then, start acquisition of the full plate.

For the end point assay use the DAPI and GFP channels to image the full plate. To run an image analysis on stained cells in the end point assay, after calculating a shading model, according to the text protocol under View output settings select the input folder with the images and an output folder with the results. Then, define the text that the images have in common, and load the shading models.

Click Start test mode and make sure the pause and displays setting for all modules are disabled. Enable the pause and display setting for module nine ImageMath. The enabled pause symbol will appear in yellow.

The enabled display function will appear as an open eye. Next, click Run to run the analysis up to module nine. In module nine adjust the parameter multiply the second image by to a value that will suppress the DAPI signal in Brucella without suppressing the nuclei.

To test the values for the module press Step. Right click on the newly opened image and set image contrast to log normalized. Repeatedly, step back to module nine.

Adjust the parameter multiply the second image by and step over the module again until the Brucella signal is fully suppressed while at the same time black areas that indicate oversubtraction are minimized. Finding good parameters for certain modules requires gradually optimizing the parameters. It is definitely important to repeatedly step over the modules, trying different values and observe the effect on the displayed result.

Enable pause and display for module 10. Identify primary objects. Set the parameter lower bound on threshold, high enough so that nuclei are segmented and background is ignored.

To identify a good value step over the module then right click the input image and display the histogram where the peak typically indicates background. Starting from background intensity increase the parameter until the good segmentation of nuclei is achieved as displayed in the output image. The goal of the image analysis pipeline is to identify cells which contain replicating bacteria.

This is achieved by setting different thresholds for the pathogen signal in the nucleus, peri-nucleus, and Voronoi cell body. Enable pause and display for module 15. Filter objects, as shown before and press Run.

Set the parameter minimum value sufficiently high so that only cells with clearly visible Brucella in the nucleus are kept, and all others are filtered away. To identify a good value step over the module and display the output image. Increase the value stepwise until only cells with clearly visible Brucella at the nucleus are kept.

After filtering for cells with bacteria in the peri-nucleus and Voronoi cell body in modules 16 and 17 according to the text protocol, exit the test mode and uncheck all the eye symbols next to the modules to make sure that none of the modules show their display prior to running the full analysis. Finally, press Analyze Images to start the analysis. When the analysis has finished inspect the resulting PNG images as well as the CSV spreadsheet to ensure that the analysis worked reliably throughout the plate.

Carry out the entry assay and infection scoring according to the text protocol. This figure shows an example of image analysis used to automatically identify infected cells in the end point assay. Nuclei HeLa cells stained with DAPI were identified.

A peri-nucleus of eight pixels width surrounding the nucleus. And a Voronoi cell body by extension of the nucleus, by 25 pixels, were calculated. A cell was considered infected if the pathogen signal exceeded the corresponding threshold in at least one compartment.

Brucella requires acting rearrangements for successful engagement of host cells. Thus, depletion of ARP23 is a suitable positive control for siRNA screening. Depletion of ARPC3 reduced the number of cells with proliferating bacteria two days after infection.

Compared to scrambled treated controlled cells. Automated image analysis quantifiably observed to decrease in infection of ARPC3 depleted HeLa cells compared to controlled cells in this graph. Depletion of ARPC3 also showed a reduction in Brucella infection in the entry assay compared to controlled cells.

As seen by the reduced number of intracellular Brucella depicted in yellow. Finally, quantification of the infection by automated image analysis confirmed that the number of infected cells as well as the number of intracellular bacteria in infected cells was reduced by depletion of ARPC3. Once mastered, this protocol can give image analysis results within one and a half weeks after starting the experiment.

While attempting this procedure it's important to carefully review the image analysis results to ensure that the chosen parameters perform well throughout the full plate. Following this procedure other methods like life cell imaging can be performed in order to answer additional questions. Like when you are trafficking a certain host factor interacts with Brucella.

Or how depletion of a gene affects intracellular dynamics of infection. After it's development, this technique paved the way for researchers in the field of infection biology to explore host factors required for Brucella infection in a HeLa cell assay. After watching this video you should have a good understanding of how to identify host factors which promote, or prevent, the successful colonization of HeLa cells by the intracellular pathogen Brucella abortus.

Don't forget that working with Brucella abortus can be extremely hazardous and local biosafety regulations have to be strictly followed.