The overall goal of this procedure is to visualize and quantitate chlamydial inclusion growth dynamics in living cells. This is accomplished by first generating fluorescent host cells that stably express a fluorescent protein such as GFP in their cytoplasm. Next, the host cells are infected with chlamydia and allowed to incubate.
Then the infected cells are visualized by live fluorescence microscopy. Finally, chlamydia inclusion characteristics are quantified through microscope analysis software ultimately live fluorescence microscopy is used to visualize chlamydial inclusions within the host cell and to measure biological parameters of inclusions for quantitative results. The main advantages of this technique over existing methods like antibody-based enumeration are that this reverse imaging strategy can be used on live cells and also in collaboration with other fluorescent labels to quantitate inclusion dynamics of all chlamydial species.
Furthermore, unlike routine methods that label bacteria in cells, this approach illuminates the inclusion membrane in living cells and can thus reveal important dynamic properties of this pathogen containing compartment. After generating fluorescent hela cells, according to the text protocol plate, GFP Hela cells on a desired culture vessel, such as glass bottom dishes for high resolution imaging 4 24 well plates for IFU determination and multi-well screening thaw a frozen tube containing crich cotus, or any other desired chlamydial strain on ice and dilute in HBSS to the desired multiplicity of infection. To perform a static infection with Crich Cotus, LGV zero VL two aspirate the media from the wells and use HBSS to wash the GFP hela cells.
Add diluted bacteria to the wells and incubate at 25 degrees Celsius for two hours, aspirate the cells and wash with HBSS. Then after adding growth medium, incubate the cells at 37 degrees Celsius in a carbon dioxide incubator to perform a centrifugation aided infection with crich to serovar D or any other chlamydial strain. Use HBSS to wash the GFP hela cells and add diluted bacteria to the cells.
Then place the dish of infected cells in a multi-well plate holder of a swinging bucket rotor in a benchtop centrifuge. Centrifuge the cells at 900 times G and 25 degrees Celsius for one hour. Then place the culture vessels in a 37 degree Celsius carbon dioxide incubator for one hour in a biosafety cabinet, aspirate the cells wash twice with HBSS and add growth medium.
Incubate the dish of infected cells at 37 degrees Celsius. To visualize chlamydia inclusions, remove chlamydia infected GFP hela cells from the carbon dioxide incubator and use RPMI without phenol red and 5%FBS To replace the media, place the dish or multi-well plate on an inverted fluorescence microscope with a 20 x or higher oil objective. Using imaging software, identify chlamydia infected GFP hela cells that appear as green cells containing large black holes.
In the acquisition setup dialogue box, configure the software to acquire the green channel and any other channel added in the sample. Use the capture slash record button to acquire the image at the desired times.Post-infection. Remove chlamydia infected GFP hela cells from the incubator and place the dish on an inverter fluorescence microscope.
Focus on the mid plains of the cells where inclusions are at their maximum diameter and yield. Crisp edges acquire images in at least 10 fields per well. Wells typically represent replicates or experimental variables.
To find GFP hela cells under the measurements tab, use the find objects command and select the green channel. Adjust the threshold intensity based on relative intensities of cells. Manually count the number of inclusions or black compartments and GFP hela cells by eye or use imaging software algorithms to automatically identify and count inclusions.
Filter the selected objects by keeping only those greater than five square micrometers. Refer to this as population one. Using this population, apply the fill holes in objects command as the input this step generates population two.
Subtract population one from population two to yield positively marked chlamydia inclusions designated as population three. Use the filter population command to apply a size filter to population three. For example, keeping objects greater than 25 square micrometers for inclusions in cells, infected for 24 hours or longer for early infection times set the size filter to keep objects greater than foursquare micrometers.
As these inclusions are much smaller, the size filter results in a population of objects designated as inclusions. Use the imaging software to calculate quantitative data from the images such as inclusion number, inclusion, size, and inclusion. Circumference upon infection with chlamydia inclusions are readily visible as black spots and the host cells or fluorescent protein expressing chlamydia within host cells, and their clarity can be exploited for automated identification across numerous fields of view and or treatments.
As demonstrated in this graph, one major application is the determination of chlamydia inclusion forming units in live infected cells, which obviates the need for immunofluorescence procedures, which are both time consuming and costly. Another key feature of this imaging approach is that it can be readily applied to any chlamydia species or strain. In this example, a time course analysis of GFP hela cells infected with one of four chlamydial species or strains was performed for each of these species and strains.
Inclusions were marked and analyzed to calculate their average area perimeter length and number per cell at times indicated. These attributes provide important information about the biology of chlamydial inclusions, including how they interact with the host cell. Shown here is a video of m cherry hela cells infected with GFPC, Tracom L two composed of time-lapse images taken at five minute intervals for 125 minutes, beginning at 48 hours post-infection.
After watching this video, you should have a good understanding of how to use this reverse imaging technique to not only visualize Chlamydial inclusions, but to quantitate inclusion dynamics such as inclusion area perimeter, and number of inclusions per cell. This technique is cost-effective compared to more traditional visualization techniques and works with all chlamydial species and strains.
