The aim of this procedure is to visualize responses of immune cells to bacterial toxins. First, the toxin is expressed in and purified from BL 21 gold cells. Once toxin activity and concentration are confirmed, the toxin is delivered to immune cells.
Using a micro injector and high speed live cell microscopy is performed. Analysis of the resulting images reveals the realtime kinetics of immune cell responses to the toxin. This method can help answer key questions in the field of immunology, such as the mechanism of inflammasome activation.
Though this method can provide insight into toxin mediated immune cell responses, it can also be applied to other stimuli including chemotactic tractors. We first have the idea for this method when studying the interaction of bacteria with cultured immune cells and observed highly variable interactions between bacteria and the immune cells. Begin the purification of strep lysin O or SLO with a culture of BL 21 gold cells containing PAG three SLO.
His plasmid grown to an OD of approximately 0.6. To induce protein expression, add five milliliters of 20%of OSes to the culture and shake at 2 25 RPM at room temperature for three hours. Next, transfer the bacteria to a 500 milliliter centrifuge bottle.
Then spin the culture at 12, 000 times G for 12 minutes of four degrees Celsius following the spin decant. The supinate store the pellet at minus 80 degrees Celsius overnight or longer if needed. To purify the expressed SLO add 10 milliliters ly wash buffer supplemented with Triton X 100 lysozyme and phenyl methyl sulfuryl fluoride to the frozen pellet pipette up and down for about 15 minutes.
To Resus, suspend the pellet keeping the sample on ice. Transfer the resuspended pellet to a 50 milliliter oak ridge round bottom polypropylene tube. Sonicate the lysate using a probe sonicate at 40%output five times for 30 seconds each at 30 seconds intervals with 30 seconds on ice between.
Then spin the sonicate lysate at 39, 000 times G for 20 minutes of four degrees Celsius. After the spin, add the bacterial supinate to one milliliter of washed nickel NTA aros. Incubate the nickel NTA aros and lysate together for 2.5 hours at four degrees Celsius with gentle shaking After the incubation.
Pellet the beads by spinning at 400 times G for five minutes at four degrees Celsius. Note that all further washes and elution are performed with these centrifugation settings and less otherwise noted. Following the centrifugation.
Combine 30 microliters of the supinate with 10 microliters four times SDS sample buffer in a micro centrifuge tube and save it on ice for purity analysis. Then discard the remainder of the supine agent and wash the beads four times in lice wash buffer. And once interest salt buffer centrifuging between each wash From this point forward, be sure to keep the sample on ice at all times.
SLO is a very redox sensitive protein and it will rapidly lose its activity if warm to 37 degrees centigrade, even if for a short period of time. To elute the SLO protein, add one milliliter of elution buffer and five microliters of one molar DTT to the beads, incubate on ice for 10 minutes. Then centrifuge and collect the supinate in a fuge tube labeled with the elucian number.
Repeat this process three times then to deplete endotoxin from the SLO protein, add 200 microliters of washed poly mixing conjugated agro beads, suspended res salt buffer to the sample and shake at four degrees Celsius for 30 minutes. Then spin at 10, 000 times G for one minute at four degrees Celsius. After the spin, collect the supernatant in new labeled micro centrifuge tubes.
Combine 30 microliters of each elution with 10 microliters, four times SDS sample buffer. For SDS page analysis, store the SLO solutions on ice. Determine protein concentrations and hemolytic activity if satisfactory, pull the SLOE in five to 10 microliter quats, then freeze on dry ice and store at minus 80 degrees Celsius.
Then determine the specific lysis.Reese. Suspend the cells to be tested at two times. Center the six cells per milliliter in buffer RB with 20 micrograms per milliliter.
Propidium iodide. Here T 27 A cells are tested. Add 100 microliters of cells to each well of a 96 well V bottom plate in separate micro centrifuge tubes.
Serially dilute SLO to two times the final concentration in buffer rb. Then add either 100 microliters of toxin or 100 microliters of buffer RB to the cells and incubate for five minutes of 37 degrees Celsius. Typical final concentrations range from 2000 units per milliliter to 31.25 units per milliliter.
Run cells on a flow cytometer and collect data with filters for fi, cethrin or pe. A one log shift represents transiently permeable cells while a three log shift indicates dead cells. Calculate the specific lysis of the cells by subtracting the percentage of dead cells in the control from the experimental using the equation shown here one day before the experiments plate, two times center the five macrophages on collagen coated glass.
Bottom 35 millimeter dishes. The microscope used for toxin delivery should be equipped with an inverted stage, a heated stage, the appropriate excitation emission filter cubes, and if available a bertand lens. The most difficult part of this procedure is bringing the needle intact down to the cells.
To ensure success, we use a Bertrand lens, which is normally used for alignment to visualize the needle as we bring it through the focal planes. The microscope should be connected to a micro injector and should be controlled by a computer with sufficient memory to collect and store data, turn on the microscope and micro injector and allow the heated stage time to warm to 37 degrees Celsius while waiting for the stage to heat. Label the cells for 30 minutes with dye at 37 degrees Celsius.
Depending on the assay labeling may be done with five microliters, four or 2:00 AM in one milliliter, HBSS or two microliter calcium am in one milliliter full medium here, four or two is used. Remove the cell medium and wash the cells once with PBS. Aspirate the PBS and replace it with one milliliter RPMI supplemented with two millimolar calcium chloride.
Mount the dish on the microscope, bring cells into focus with brightfield, then adjust to focus slightly above the cells. Next, combine one microliter of SLO toxin four microliters, 10 milligram per milliliter, DEXTRA 5 55 and six microliters of water. Then centrifuge your 20, 000 times G for 10 minutes of four degrees Celsius.
Dextran is used to verify that the femto tip is not G clogged. Use a micro loader to load two microliters of diluted toxin into the femto tip from the rear. Load the femto tip onto the micro injector.
Then adjust the angle of the injector so that the tip will sit over the center of the cells with room to move in all directions clear the previous settings for Z limit which limits how low the micro injector tip can go. Set the micro injector to inject for 0.5 seconds at 120 PSI with 20 PSI back pressure. Then lower the tip until it enters the medium.
Using the Bertrand lens. Center the tip and follow the tip as it is lowered closer to the cells. Once the needle goes outta focus, switch back to normal optics.
The needle shadow should be apparent in the field. Focus above the cells and lower the needle until it comes into focus. Then bring the cells into focus and carefully bring the needle adjacent to a cell.
After setting the Z limit for injection, begin imaging and inject toxin. Then raise the needle and move to a new region of cells. Inject additional toxin and continue imaging following the injection.
Move the needle to the home position to prevent any undesired toxin leakage from the needle using the method described in this video, 10 to the seven to 10 to the eight units per milliliter. SLO is typically obtained with a protein concentration of four milligrams per milliliter. This SDS page gel shows bacterial samples before and after toxin induction sate following purification, and each of three ellucians kamasi blue staining reveals that the induced SLO was successfully purified.
SLO is the band at 69 kilodaltons to determine the amount of toxin required for lysis of T 27 A or D two cells. Cells were challenged with various concentrations of SLO for five minutes at 37 degrees Celsius in the presence of propidium iodide and examined by flow cytometry. Specific lysis was determined as demonstrated as shown here.
250 units per milliliter. SLO gives 50%lysis of both T 27 A and D two cells. A sub litic 10%lysis dose of toxin would be 62.5 units per milliliter.
These values are important for benchmark toxin activity to assess cell death following toxin exposure. Human dermal fibroblasts were incubated with calcium and atherium homodimer using a live dead kit from life technologies. Following localized exposure to the bacterial toxin, anthro lysin O.In this image, calcium is shown in green and atherium bromide in red regions close to the microbiome, show cell death typified by calcium loss and athe homodimer uptake, whereas regions further away from the tip will show no damage.
Micro delivery of the toxin also allows the examination of real-time events such as calcium flux. These human dcs loaded with 4:02 AM Were exposed to SLO at a constant flow rate with simultaneous live imaging shift from green to blue. Pseudo color indicates an increase in cytosolic calcium levels.
SLO induces a rapid increase in cytosolic calcium, which is propagated through the region of the dish due to the localized delivery. However, only cells close to the micro injector tip encounter and reactor toxin. This demonstrates both one cellular reaction to toxin and the spatially restricted area of toxin delivery to resolve events in the zdi dimension.
Micro delivery was combined with high speed 3D confocal microscopy. This set of images shows dendritic cells following toxin injection. They were pre incubated with anti CD 11 C conjugated to a PC shown in green to label the plasma membrane.
As can be seen here, microvesicles are released following toxin delivery. As part of the cellular repair process, toxin molecules are concentrated on blebs, which is shed to eliminate toxin. Once master, the microscopy can be done in 45 minutes.
While attempting this procedure, it's important to remember to keep the toxin cold prior to mixing with cells and to test the hemolytic activity.Good. After watching this video, you should have a good understanding of how to measure real time kinetics of immune cell responses to bacterial toxins using live cell microscopy.
