This protocol provides a multifaceted understanding of the activation of cell death processes, which can provide critical insights into disease mechanisms and inform therapeutic strategies. This protocol relies on the use of a simpler technique and to access the activation of multiple caspases imperative from single population of endogenous cells to greatly determine activation. Innate immune cell death has been implicated across the disease spectrum, from infections to inflammatory diseases to cancers.
Understanding the molecular mechanisms of cell death through caspase activation can provide critical insights into disease processes. Dr.Julieann, a post-doctoral research associate from a laboratory, will be demonstrating the procedure. After isolating the bone marrow and differentiating the BMDMs, infect the cells with the influenza A virus.
Calculate the virus volume needed at a multiplicity of infection of 20 plaque-forming units. Remove the media from the BMDMs and wash the cells once with 500 microliters of PBS. Add 450 microliters of Influenza A virus in high glucose DMEM without heat inactivated FBS to each well, and incubate the plates at 37 degrees Celsius for one hour in a humidified incubator to allow absorption.
At the end of the one-hour incubation, add 50 microliters of heat inactivated FBS and return the plates to the incubator at 37 degrees Celsius for a total of 12 hours. After 12 hours of incubation, remove the plate from the incubator. Aspirate 150 microliters of the supernatant and discard or save this for supernatant analysis.
Do not remove the remaining supernatant. Now, create the protein collection solution by combining 50 microliters of caspase lysis buffer and 100 microliters of four XSDS buffer per well. Then, add 150 microliters of the mix to each well.
For each well, pipette the mixture to collect the lysed cells and supernatant. While pipetting, scrape the bottom of the well with the pipette tip to disrupt the cells. After scraping and pipetting, collect the protein lysate into labeled 1.5 milliliter tubes.
Using a heat block, heat all the tubes to 100 degrees Celsius for 12 minutes. Remove the tubes from the heat block and centrifuge at 14, 500 G for 30 seconds at room temperature to pellet any insoluble components. Prepare the electrophoresis apparatus with a 12%polyacrylamide gel with 10 wells.
Fill the electrophoresis apparatus with running buffer and remove the gel comb. Then, heat the samples at 100 degrees Celsius for five minutes. Centrifuge the samples at 14, 500 G for 30 seconds at room temperature before loading.
Then, slowly load 30 microliters of the sample into each well. Use combined supernatant and protein lysate and caspase lysis buffer or the tissue homogenate for caspase 1, 3, 7 and 8 blots, and use the protein lysate DN RIPA buffer described in the protocol or the tissue homogenate for caspase 11 and 9. To evaluate all six caspases at once, use the same procedure to load the same samples into each of the six gels.
Connect the electrophoresis apparatus to the power source and set the power to 80 volts for 20 minutes to begin the gel run. After the initial 20 minutes, adjust the power to 100 volts for 45 to 60 minutes. Observe the dye front.
Once the dye front reaches the bottom of the gel, turn the power off. While the gel runs, prepare the transfer buffer as described in the manuscript. Make the solution fresh each time.
Remove the gel from the electrophoresis apparatus using the gel releaser. To set up the transfer stack for the gel, activate a PVDF membrane by soaking it in methanol for one minute. Pre-wet two pieces of filter paper, the gel, and the PVDF membrane and transfer buffer for five minutes.
Keep the PVDF membrane and gel in separate containers during this five-minute incubation. Begin assembling the transfer stack on the semi-dry system. On the bottom platinum nano side, place one piece of filter paper, the PVDF membrane, the gel, and finally, one piece of filter paper.
Gently roll out or press out air bubbles between the layers and close the top of the system. Now, connect to the power source. Set the power to 25 volts for 40 minutes.
After the transfer, disassemble the transfer stack, collect the membrane, and place it in a square Petri dish. Next, perform membrane blocking by adding 15 milliliters of a 5%skim milk solution and incubate the membrane on a rocking shaker at 50 to 70 RPM at room temperature for one hour. After incubation, remove the blocking solution, add 10 milliliters of the diluted antibody solution, and incubate for two hours at room temperature or four degrees Celsius overnight on a rocking shaker, as demonstrated previously.
Then, collect the antibody solution and wash the membrane by adding 15 milliliters of TBST to the membrane on a rocking shaker at room temperature for 10 minutes. Discard the TBST. Repeat the wash with 15 milliliters of TBST three times.
Add 10 milliliters of the diluted secondary HRP conjugated antibody solution. Incubate on a rocking shaker at room temperature for one hour. At the end of the incubation, once the antibody solution is removed, wash the membrane by adding 15 milliliters of TBST on a rocking shaker at room temperature for 10 minutes.
After completing the washing steps and removing the TBST, add 10 milliliters of the high sensitivity HRP substrate. Let it sit at room temperature for one minute. Remove the membrane from the substrate.
Proceed directly to the imaging using a chemiluminescence imager with the accessory white trans tray inserted in the lower position. Expose the membrane using the auto exposure mode. The pro and activated caspase 1, 11, 3, 7, 8 and 9 in wild type and ZBP1 mutant after Influenza A virus infection, wild type and AIM2 mutant after HSV1 infection, and Francisella novicida infection, or wild type and NLRP3 mutant BDMs after lipopolysaccharide and ATP stimulation are analyzed.
Cells lacking upstream PANoptosis sensors do not undergo robust cell death in response to the cognate PANoptosis-inducing stimuli. Influenza A virus infection induces the formation of the ZBP1-PANoptosome and the ZBP1 deficient cells are significantly protected from cell death during Influenza A virus infection. Similarly, HSV1 and Francisella novicida infections induce the formation of the AIM2 PANoptosome, and AIM2 deficient cells fail to undergo robust cell death in response to these infections.
Cells lacking upstream inflammasome sensors are protected from cell death in response to their respective stimuli, and NLRP3 deficient cells do not undergo cell death in response to lipopolysaccharide and ATP. It is important to remember to prepare a mixture of both cell lysate to determine a specific caspase. After loading the jet, the luminous sample should be stored minus 20 degree to maintain integrity of the goals of caspase for following up In conjunction with this protocol, real-time cell death imaging or LDH assays can be used to monitor the execution of cell death, and ELIZAs can be used to check for the release of cytokines or DAMPs from cells undergoing different types of cell death.
