This protocol allows researchers to identify if caspase activity occurs in conjunction with cell death, thus identifying different modes of cell death. The advantage of this technique is the flexibility to assess caspase activity at a population-based assay or single cell. Demonstrating the procedure will be Stefanie Rufli, a PhD graduate, and Jing Tong, a visiting PhD student from the laboratory of Wendy Wei-Lynn Wong.
After differentiating and harvesting the bone marrow-derived macrophages or BMDMs as described in the manuscript, seed them in a six-well plate at the density of one times 10 to the sixth cells per milliliter. Treat the cells with SMAC mimetic for 16 hours. To prepare the cell lysate, transfer the plate containing treated cells onto the ice and collect the cell culture medium into a 1.5 milliliter tube.
Centrifuge at 300 G for five minutes at four degree Celsius. Aspirate the medium and put the tube on ice. Then add one milliliter of cold PBS to the cell culture plate to wash the cells.
After aspirating all the PBS, add 100 microliters of trypsin to the cells. Once the cells are detached from the plate, collect them into the 1.5 milliliter tube. After centrifuging the cell suspension, remove the supernatant and resuspend the pellet in 100 microliters DISC lysis buffer.
Incubate the sample on ice for 20 minutes and then centrifuge the lysate to pellet the insoluble fraction. Transfer 25 microliters of lysate to a white flat-bottomed 96-well plate for the caspase-3/7 activity assay and 10 microliters to a transparent flat-bottomed 96-well plate for the bicinchoninic acid or BCA assay. For protein quantification, prepare a range of bovine serum albumin or BSA concentrations as a standard protein.
Once standard BSA is added to the flat-bottomed 96-well plate containing the samples, mix the BCA reagents one and two at a ratio of 50 to one and add 200 microliters of it to each sample in standard. After incubating at 37 degrees Celsius for 30 minutes, measure the absorbance at 562 nanometers on a fluorometric instrument and quantify the protein concentration with the standard curve. To perform a population based-assay, start the fluorometric instrument and heat the machine to 30 degrees Celsius.
Set the excitation and emission wavelength at 360 and 465 nanometers respectively. Then prepare a master reaction mix on ice as described in the manuscript to determine the caspase activity. Add 75 microliters of the mix to each sample and standard to obtain a total reaction volume of 100 microliters.
Immediately measure the fluorescence using the fluorometric instrument setup and record individual readings every minute for 40 minutes to determine the reaction kinetics. After seeding, harvest the adherent cells as demonstrated previously into a five milliliter polystyrene tube. Then centrifuge the sample at 300 G for five minutes at four degrees Celsius, and remove the supernatant.
To prepare the staining mix, take one microliter of the fluorogenic substrate and dilute it with 150 microliters of PBS. Add 50 microliters of staining mix per sample, incubate it in the dark at 37 degrees Celsius for 30 minutes and mix every 15 minutes. In this population-based assay, the representative data of the kinetic assay for caspase-3/7 activity showed increased substrate cleavage with increased SMAC mimetic concentration.
However, the increase at 500 nanomolar concentration was insignificant based on an ordinary one-way ANOVA with multiple comparisons. Analysis of caspase-3/7 activity using flow cytometry indicated a shift in fluorescence for the cells treated with SMAC mimetics compared to the untreated cells, which was also evident in the plotted median fluorescence intensity and the increase at 500 nanomolar concentration was significant. The procedure should be performed on ice.
However, in population-based assay, samples cannot be left on ice indefinitely. After the lysis step, samples should be further processed or frozen immediately. Imaging the caspase activity using live cell microscopy would be an additional method to gain kinetic information on a single cell level.
