The overall goal of this procedure is to measure the effect of phenotype modulators such as host gene silencing or the impact of small molecules on mycobacterium tuberculosis, intracellular replication. This is accomplished by first dispensing small molecules and 384 well plates or transecting cells with irna and transfect into complex. The next step is to infect cells with green fluorescent protein expressing mycobacterium tuberculosis or G-F-P-M-T-B add cells to the small molecule containing wells and incubate the microplate for five days.
For the RNAi approach, add cells onto RNAi transfect complex containing wells. Incubate the microplate for three days and then infect cells with G-F-P-M-T-B expressing mycobacterium tuberculosis. After staining the cells, the final step is to read the plates using an automated confocal microscope.
Ultimately automated fluorescence microscopy followed by image-based analysis is used to measure the changes in G-F-P-M-T-V intracellular growth. The main advantage of this technique of existing method like colony forming uni hunting is that it spare long incubation period and allows more throughput. Demonstrating this procedure will be Christophe Keval or SONG and three postdoctoral fellows from my research team.
The protocols for SI RNA library screening and compounds library screening utilize two week old GFP expressing MYCOBACTERIUM tuberculosis H 37 RV cultures. To prepare the G-F-P-M-T-B bacterial suspension first centrifuge the culture at 4, 000 Gs for five minutes, discard the supernatants Resus, suspend the culture into DPBS and centrifuge again in this manner. Wash the culture three times with DPBS.
After the third wash, discard the supernat and resuspend the bacterial pellet in 10 milliliters of 10%FBS containing RPMI 1640 medium. Leave the suspension for one hour at room temperature to allow the bacterial aggregates to sediment. Collect the bacterial supernat and measure the OD at 600 nanometers and GFP fluorescence.
Using a microplate reader to determine the bacterial concentration, the OD at 600 nanometers should be between 0.6 and 0.8. Calculate the titer of the suspension using a reference regression line, displaying RFU value as a function of CFU value generated prior to the experiments on another culture that was prepared in the same conditions. A typical concentration is one times 10 to the eight bacteria per milliliter.
Begin this procedure by Resus suspending the dried SI RNA library that is stored in 96. Well mother plates with one X-S-I-R-N-A buffer to a concentration of two micromolar. Transfer 10 microliters of the mixture into each well of a 384 well daughter plate transfer 2.5 microliters of SI RNA from each well of the daughter plate into a 384 well assay plate to the same assay plate.
Add 2.5 microliters each of a negative and positive control IRNA to their respective wells. If the daughter plate will not be used immediately seal it with a peelable aluminum seal. The sealed plate can be stored at negative 20 degrees Celsius for at least six months and possibly up to two to three years depending on the IRNA.
Library manufacturer's recommendations. Prepare the transfection reagents by diluting it in one X-D-P-B-S to yield enough solution to provide 0.1 microliters for each. Well pre incubate the diluted transfection solution at room temperature for five minutes.
Add 7.5 microliters of the diluted transfection solution to each well of the 384 well assay plate and incubate for 30 minutes at room temperature to each well of the assay plate at 40 microliters of human type two pneumocytes, A 5 4 9 cells suspended in RPMI 1640 medium supplemented with 10%fetal bovine serum. Maintain cells for a three day incubation period at 37 degrees Celsius in an atmosphere containing 5%carbon dioxide. These cells divide every 24 hours, thus about 12, 000 cells are in each well three days after transfection.
Three days after si. RNA transfection of a 5 49 cells prepare an MTBH 37 RV GFP bacterial suspension. As demonstrated earlier.
The suspension should contain 2.4 times 10 to the six bacteria per milliliter, which corresponds to a multiplicity of infection of five. Remove the medium in the 384 well assay plate and add 25 microliters of the bacterial suspension per well. Incubate the 384 well assay plate at 37 degrees Celsius for five hours in an atmosphere containing 5%carbon dioxide.
After five hours. Remove the medium and gently wash the cells three times with RPMI medium supplemented with 10%FBS to kill the remaining extracellular bacteria. Treat the cells in each well with 50 microliters.
A fresh R-P-M-I-F-B-S medium containing 50 micrograms per milliliter of Amikacin. Incubate at 37 degrees Celsius for one hour in an atmosphere containing 5%carbon dioxide. Lastly, remove the medium containing Amikacin and add 50 microliters of fresh RPMI medium supplemented with 10%FBS per.Well.
Incubate the assay plate at 37 degrees Celsius for five days in an atmosphere containing 5%carbon dioxide before screening by confocal microscopy. To begin this procedure thaw a 384 well mother plate containing the compounds library solubilized in 100%DMSO at room temperature transfer 0.5 microliters of the compounds from the mother plate to a 384 well daughter plate containing 10 microliters per well of RPMI 1640. Medium supplemented with 10%FPS next harvest six day old primary human macrophages at four times 10 to the five cells per milliliter in RPMI 1640.
Medium supplemented with 10%FPS and 50 nanograms per milliliter. Recombinant human MCSF incubate the diluted primary cells with basia at different MOIs ranging from one to five in suspension with mild shaking at 90 RPM for two hours at 37 degrees Celsius. Wash the infected cells by centrifugation at 350 GS for five minutes to remove the extracellular bacteria.
Resus suspend the pellets in RPMI 1640. Medium supplemented with 10%FPS and centrifuge. Again, repeat this, wash twice after the final wash.
Resus suspend the infected cells in RPMI 1640 medium supplemented with 10%FBS and 50 micrograms per milliliter. Amikacin incubate the suspension with mild shaking for one hour at 37 degrees Celsius centrifuge at 350 GS for five minutes. Remove the medium containing Amikacin and wash the infected cells with complete RPMI 1640.
Medium supplemented with 10%FBS and 50 nanograms per milliliter. Recombinant human MCSF. Repeat this wash once.
Add 40 microliters per well of the infected macrophage suspension to the same 384 well assay plate prepared earlier, which already contains 10 microliters of the compound's dilutions. The final concentration of DMSO in each well is now 1%Incubate the assay plates for five days at 37 degrees Celsius in an atmosphere containing 5%carbon dioxide before screening by confocal microscopy for SI RNA library. Screening prior to image acquisition, add to each well of the assay plate 10 microliters of freshly prepared 30 micrograms per milliliter DPI in PBS and incubate for 10 minutes at 37 degrees Celsius.
For compound screening Prior to image acquisition, stain the live cells with cell permeant far red fluorescent dye for 30 minutes at 37 degrees Celsius. Load the assay plates into an automated confocal microscope. Set the exposure parameters record DPI fluorescence using an excitation laser at 405 nanometers with an emission filter at 450 nanometers.
Record GFP fluorescence using an excitation laser at 488 nanometers with an emission filter at 520 nanometers. Select the wells and the fields at each well to be acquired, which are then referred to as layouts and sub layouts. Parameters generate the experiments file using the set parameters and run the automatic acquisition.
In this case, four different images of the same well and field are recorded transfer images to a remote server. Subsequently the images are evaluated using the acapella 2.6 image analysis software for SI RNA screening. Each field contains two channels or colors.
Green for the bacteria and blue for the cell. Nuclei detect cell nuclei from the DAPI channel using a nuclei detection algorithm and the bacterial area from the GFP channel using a pixel intensity properties algorithm. By merging the channels and counting the number of pixel shared by both bacteria and cells, the intracellular bacteria is quantified.
The final results expressed as an average of the four fields are the total bacterial area, the total number of cells, the percentage of infected cells, and the bacterial area per cell. For compound screening, each field contains two channels or colors, green for the bacteria and far red for the cell. Nuclei and cytoplasm detect the cell area from the far red channel and the bacterial area from the GFP channel using a pixel intensity properties algorithm.
By merging the channels and counting the number of pixels shared by both bacteria and nuclei, the intracellular bacteria is quantified. The final results expressed as an average of the four fields are the total bacterial area, total cellular area, total area of intracellular bacteria, and the ratio between intracellular bacterial area and total area of cells. Representative results from the high throughput genome-wide SI RNA screening are presented here, silencing the expression of din one with SI RNA led to a decrease of intracellular mycobacteria amount in a 5 49 cells.
As shown in these representative confocal images of a 5 49 cells transfected with non-target IRNA or with SI RNA specific for din one and infected with GFP expressing MTB for five days. GFP MT BH 37 RV was visualized in green and the cells in red. The number of cells and the intracellular G-F-P-M-T-B-H 37 RV load were determined using image-based analysis software.
This graph shows the percentage of infected a 5 49 cells in five replicates of scramble IRNA represented by blue circles and din one irna represented by red circles. After three days of silencing and five days of infection, the percentage of infected cells is reduced by half in din one silenced a 5 49 cells compared to cells transfected with non-target scramble IRNA. A sample based normalization of SI RNA targeted DIN one compared to scramble was applied to define the Z-score.
A Z-score average arounds negative 15 was obtained for SI a targeted DIN one. The three asterisk represent a P value of less than 0.0001. These results indicates that DIN one can be used as a positive control for the si, a screen to discover other novel host factors involved in MTB colonization and pneumocytes that could have the same phenotype as that with dins IRNA in the high contents compound screening.
Compound efficiency on intracellular bacterial growth was evaluated by establishing a dose response curve or DRC and normalizing to the reference positive and negative compounds. In this example, human primary macrophages infected by A GFP expressing MTBH 37 RV strain were incubated with 1%DMSO as a negative control or with increasing concentrations of two reference compounds, isid, INH and rifampicin.RIF. The macrophages are labeled with a red fluorescence dye and the green color indicates the MTB analysis of the confocal fluorescence images of the infected human macrophages revealed that the active compounds impact the intracellular replication of MTB in host cells leading to a decrease of mycobacterial load, which corresponds to the area of the GFP signal in cells.
The ratio between the intracellular bacterial area and total cell area was calculated and then plotted as a function of compounds concentration to generate the DRC shown here are the DRCs of isid and rifampicin in each graph. The DRC of the compound was normalized to that of 1%DMSO, the negative control and 0.1 micrograms per milliliter isid the positive control. These curves allow the determination of both the concentration required to inhibit 50%of the bacterial colonization and the minimal concentration required to inhibit 99%of bacterial replication One semester.
This, this technique can be done in 10 hours divided as follows, two hours for cell transfection or compound preparation, six hours for cell infection and dispensing into microplate and two hours for sustaining an image acquisition. This over an eight day period, don't forget that working with mycobacterium tuberculosis can be extremely as artists and that precocious, such as wearing personal protective equipment, including a mask, should always be taken while performing this procedure.
