This protocol is useful for determining changes in NF-kappa-B activation in cells expressing an NF-kappa-B luciferase reporter. The main advantage of this technique is it allows for a high-throughput screen of factors or conditions that lead to changes in NF-kappa-B activation. NF-kappa-B is a transcription factor for a wide variety of cellular processes.
A well known function of NF-kappa-B is in the induction of inflammatory responses by inducing the expression of various cytokines and chemokines. Our lab is particularly interested in the induction of NF-kappa-B induced by the pathogen Salmonella typhimurium. Here, we use the HeLa cell line that is stably transfected with an NF-kappa-B luciferase reporter plasmid.
Other bacteria, or even viruses or chemical compounds, can be used in this cell line to study the activation of NF-kappa-B. Other cell lines can also be used if you can introduce such a NF-kappa-B luciferase reporter plasmid in that cell line. Somebody doing this for the first time may have issues properly utilizing sterile technique that is required for cell culture and bacterial work.
One day before cell stimulation, remove the growth media of HeLa 57A cells that have been grown to about 75%confluency. Wash cells with one milliliter of 0.05%trypsin EDTA. Replace with another milliliter of trypsin EDTA and transfer the flask to a 37 degrees Celsius incubator for five minutes.
After the cells have been detached from the flask, suspend them in 10 milliliters of growth media. Combine 10 microliters of cell suspension with 10 microliters of Trypan blue, pipette up and down to mix, and transfer 10 microliters to a cell counter slide. Count cells in suspension using a cell counter, and use growth media to dilute the cells to a final concentration of 2.5 times 10 to the five cells per milliliter in a 50 milliliter conical tube.
Transfer 250 microliters of the cell suspension to each well of a 48-well plate. Periodically cap and turn over the conical tube to ensure a homogenous cell suspension. Tap the plate gently on the side to ensure that the cells distribute uniformly in the wells.
Transfer the plate to 37 degrees Celsius in a 5%carbon dioxide incubator and allow the cells to attach and grow overnight. Streak frozen stocks of Salmonella onto LB agar plates to produce single colonies. Transfer the plates to an incubator set to 37 degrees Celsius and allow overnight growth.
The next day, add three milliliters of LB to sterile bacterial culture tubes, and add appropriate antibiotics to the media. Using a sterile inoculation loop, pick a single colony from the streaked bacterial cultures and touch the loop to the LB media. Cap the tubes after inoculating and discard the loop.
Place the tubes in a shaking incubator set at 37 degrees Celsius and 180 RPM, then allow the bacteria to grow overnight. In the morning, retrieve overnight bacterial cultures from the incubator. Prepare tubes for subculture by adding three milliliters of fresh LB and antibiotics.
Transfer 30 microliters of the overnight bacterial culture to the freshly prepared media. Place the tubes in a shaking incubator set at 37 degrees Celsius for three hours. After the three hour incubation, transfer one milliliter of sterile LB broth into a plastic cuvette to serve as the blank.
Transfer 900 microliters of LB into the other cuvettes to be used for the sample analysis. Transfer 100 microliters of bacterial subculture into a cuvette containing 900 microliters of LB, and pipette up and down several times to mix. Repeat this for each bacterial suspension.
Turn on the spectrophotometer to measure the optical density of the bacterial cultures at a wavelength of 600 nanometers. Place the blank in the spectrophotometer. Take note of the orientation.
Close the lid and press the blank button on the spectrophotometer to get the background absorbance. Replace the blank cuvette with a sample cuvette in the same orientation and press read. Record the OD600 values of these samples.
Multiply the value by 10 to account for the dilution factor. Dilute the suspension one to 10 in a cuvette and measure absorbance, which should give a value of approximately 0.1 which roughly corresponds to one times 10 to the nine CFU per milliliter. In a new tube, add an appropriate volume of the diluted subculture to fresh LB to achieve a suspension of one times 10 to the eight CFU per milliliter to be used as an inoculum.
Prepare serial dilutions of the inoculum by transferring 50 microliters of bacterial suspension to a tube containing 450 microliters of sterile PBS until a final dilution of approximately 100 CFU per milliliter is made. Transfer 100 microliters of the two lowest dilutions, 100 and 1000 CFU per milliliter, to two LB agar plates, and spread the suspension with a cell spreader to obtain single colonies. Transfer these plates to a 37 degree Celsius incubator and incubate overnight.
The following day, count the colonies and calculate the bacterial concentration of the initial inoculum to determine the actual inoculum concentration. On the same day, label the lid of the plate according to the infection conditions that will be used for each well, with each condition being done in triplicate. Add 10 microliters of the inoculum to appropriate wells, and add 10 microliters of sterile LB to uninfected control wells.
To synchronize the time of infection, place the plate in a tabletop centrifuge and spin at 500 times G for five minutes, ensuring that the plate is counterbalanced. Then, transfer the infected cells to a 5%carbon dioxide incubator at 37 degrees Celsius for one hour. Place a 15 milliliter conical tube containing an aliquot of cell culture media in a 37 degrees Celsius water bath for use in the next step.
One hour after the time of infection, remove the 15 milliliter conical tube containing cell culture media from the water bath and wipe the exterior with 70%ethanol. Transfer the tissue culture plates from the incubator to a biosafety cabinet. Use sterile tips to aspirate media from the wells and replace with 250 microliters of fresh, warm cell culture media.
Return the plates to the 5%carbon dioxide incubator at 37 degrees Celsius for an additional four hours. After that, remove the plates from the carbon dioxide incubator and aspirate the media from the wells. For luciferase analysis, add 100 microliters of 1X cell lysis buffer to the wells.
Transfer the plate to a negative 80 degrees Celsius freezer and incubate for at least 30 minutes to ensure efficient cell lysis. Then, place the plate containing frozen cell lysate on a bench to thaw, and prepare luciferase substrate reagents according to manufacturer recommendations. Allow luciferase substrate reagents to equilibrate to room temperature.
Next, turn on the plate reader and open the corresponding reader program. Set the machine to measure luminescence. Transfer 10 microliters of each cell lysate to a well of an opaque 96-well plate.
Using a multichannel pipette, add 50 microliters of the luciferase assay reagent to each well of the opaque plate. Gently tap the plate on the side to mix wells, and ensure that the bottom surface is covered with liquid. Place the plate in a plate reader and initiate reading.
Copy the luminescence values into a spreadsheet program and plot the results. This protocol focuses on the activation of the transcription factor NF-kappa-B using an NF-kappa-B dependent luciferase reporter that is stably transfected into a line of HeLa cells. This figure shows a representative experiment of NF-kappa-B dependent luciferase activation and IL6 gene expression in HeLA 57A cells infected with the Salmonella typhimurium strains.
Infection with the wild type SL1344 strain induced a strong luciferase signal in both the RLU and IL6 expression, which was decreased in cells infected with the sipA sopB sopE2 triple mutant and reduced to control levels with the sipA sopB sopE2 sopE quadruple mutant. Pay close attention when doing your serial dilutions and plating. This will ensure that you have a consistent inoculum across your strains.
After identifying factors that contribute to NF-kappa-B activation and downstream changes in mRNA expression, western blot technique can be used to identify changes in protein expression. This protocol has allowed our lab not only to evaluate NF-kappa-B activation induced by Salmonella but other compounds and proteins implicated in NF-kappa-B activation. Salmonella typhimurium is a human pathogen.
Employ proper PPE when working with these bacteria.
