This experiment monitors VA rupture elicited by intracellular pathogens in single cells. First load the cells with CCF 4:00 AM dye, which is cleaved by cellular esterases. Then prepare and apply the FL neuro bacteria to the CCF four loaded cells incubate the cells at 37 degrees Celsius for bacterial invasion of the host cells.
Then determine the ratio of intensities emitted in the 450 nanometer and 535 nanometer channels. Results show the presence of pathogens in the cytosol Because of its adaptability to high throughput lys. This technique facilitates the identification of novel antibiotics by addressing a key problem of bacteria subcellular localization during host pathogens Interactions.
Together with my colleague Tran Vanu, we were interested in developing approaches to look at intracellular pathogens with high temporal resolution. This led to the establishment of the CCF four betalactamase regular ular rupture assay. Today, this approach will be presented experimentally by two graduate students in the laboratory, Nora MellU and Charlotte Keller Inoculate the wild type and mutant bacterial strains in eight milliliters of TCSB containing 50 micrograms per milliliter of ampicillin place the cultures in a 37 degree Celsius shaker now seed five times 10 to the third hela cells per well.
In 100 microliters of DMEM containing 10%fetal calf serum 1%penicillin streptomycin culture, the cells in a 5%carbon dioxide incubator the next day subculture the bacteria at a one 100th dilution in TCSB supplemented with 50 micrograms per milliliter of ampicillin grow in a shaker at 37 degrees Celsius for two and a half hours. Now for loading the hela cells prepare CCF 4:00 AM dye in em buffer wash the cells once with PBS. Then add 25 microliters of CCF 4:00 AM loading mix per well at room temperature in the dark for two hours and 30 minutes to prepare the bacteria for infection pellet one milliliter of culture by centrifugation after one wash with 500 microliters of PBS Resus, suspend the bacteria in 500 microliters of PBS supplemented with 10 micrograms per milliliter of polyol lysine and 40 micrograms per milliliter of beta-lactamase.
Incubate for 10 on a rotating wheel at room temperature, then wash once with 500 microliters of PBS and resuspend each bacterial pellet in 500 microliters of one millimolar Probenecid in EM buffer wash the cells once with 150 microliters of one millimolar prob in em buffer. Next, dilute 10 microliters of bacteria in 100 microliters of one millimolar probed solution and distribute it to each well of hela cells. After 15 minutes incubation in the dark at room temperature, transfer the plate to 37 degrees Celsius for one hour.
Wash once with 150 microliters of one millimolar probit solution. Then fix samples with 50 microliters of 4%paraform aldehyde in one millimolar Pro solution for 10 minutes in the dark After one wash with Probenecid solution, add 30 microliters of 10 micromolar nuclear dye drac five for optimal cell segmentation. Incubate the cells for 30 minutes, wash once and add 100 microliters of one millimolar solution to acquire images using an inverted epi fluorescence microscope.
Use an excitation wavelength at 405 nanometers. Detect emission via 450 nanometer and 535 nanometer filters using exposure times of five milliseconds for transmitted light. 1000 milliseconds for 535 nanometer and 500 milliseconds for 450 nanometer.
The use of a focus calibration device coupled to automated microscopy allows for simultaneous acquisition of dozens of different positions in multiple wells. If using a confocal microscope, then use exposure times of 240 milliseconds at 450 nanometers, 360 milliseconds at 535 nanometers for the 405 nanometer laser and 640 milliseconds for the 640 nanometer laser. Analyze data by a computer algorithm that allows automated scoring of the fluorescent signal for each individual cell.
For example, use software such as metamorph and acapella to create a script for measuring the ratio between the 450 nanometer and 535 nanometer emission signals. For this assay, start a HELOC culture with two times 10 to the fifth cells in a final volume of two milliliters. Prepare the bacteria for infection and load the hela cells with CCF 4:00 AM arrange a convo microscope with an N plan air objective inside a 37 degrees Celsius heating chamber.
Set the 405 nanometer laser and emission via 450 nanometer and 535 nanometer filters. Also enter exposure times of five milliseconds for transmitted light. 200 milliseconds for 535 nanometer and 100 milliseconds for 450 nanometer.
Set data acquisition for every 90 seconds over 60 minutes. Now wash the cells once with two milliliters of one millimolar Probenecid solution. Add two milliliters of one millimolar prob in em buffer.
Then mount the dish on the stage of the microscope and start the acquisition after six minutes. Put the acquisition on hold. Add 250 microliters of bacteria resuspension on top of the cells and restart the acquisition for the post-acquisition analysis.
Visualize movies using Velocity metamorph or image J.Obtain 450 to 535 nanometer intensity ratios for each individual cell. The CCF 4:00 AM beta-lactamase approach is a robust and sensitive method for tracking ular rupture of intracellular pathogens such as gel flexneri upon HELOC cell infection. With the non-invasive BS 176 AFA one strain for one hour, the CCF four fret probe remains intact and emits a green signal.
By contrast, the virulent M 90 TFA I strain switches the signal towards blue consistent with probe cleavage in the cytosol for the determination of the ratio metric signal. We developed a script for the metamorph and acapella software to automate detection of the cells and measurements in the 535 and 450 nanometer channels. Nuclei and cytosol of cells are segmented using the DR five channel.
Then the algorithm is capable of detecting and quantifying the 450 nanometer and the 535 nanometer positive cell populations. The calculated mean ratios are low for the mutant strain and high for the virulent strain. Experiments can be performed on different human cell types like Gila epithelial cells and THP one macrophage like cells.
Both cell types respond to the virulent M 90 T AFA one cha strain by switching emitted signal from green to blue under infection. With the non-invasive strain, the green signal persists quantification using a script developed on metamorph software and a macro developed in Excel presents histograms of ular rupture as a function of cell distribution. The method can be successfully adapted to understand infectivity of different bacteria.
For instance, this dataset show mycobacterium Bovis. BCG resides in the phagosome for the whole course of the experiment as shown by the persistent green signal. In contrast, mycobacterium tuberculosis elicits vaga omal membrane rupture in THP one macrophages after seven days of infection as highlighted by the apparition of a 450 nanometer signal after seven days of infection using the same algorithm.
As for studying valar rupture by Ella, we found that mycobacterium tuberculosis infected cells display higher 450 to 535 nanometer ratios than mycobacterium bovis BCG after seven days of infection. After watching this video, you should have a good understanding how to study ular rupture by pathogens with the CCL four betalactamase assay Once mastered, this protocol can be performed in four hours, but remember, you have to add proce in each buffer all during the protocol. The main advantage of this technique over existing methods, such as electron microscopy or membrane fractionation, is that it can be performed in real time without any biochemical treatments.
Further analysis like actin labeling or gentamicin protection assay can be performed in order to evaluate the uptake and the proliferation of the bacteria in host cells.
