W.W.W. is supported by Cl&#246;etta Medical Research Fellow grant, S.R. is supported by the CanDoc UZH Forschungskredit, and J.T. is supported by the Chinese Scholarship Council.

The present study was performed with the approval and following the guidelines of the animal ethical committee of the University of Z&#252;rich (#ZH149/19). Male C57Bl/6J mice aged 8-16 weeks, bred and housed in specific pathogen-free (SPF) conditions, were used for the present study. The intact bones can be kept on ice in sterile Hank&#39;s buffered saline solution (HBSS) with 2% heat-inactivated fetal bovine serum (FBS). Bone marrow was collected from the femur and tibia of the mouse25 on the day of differentiation. Both methods for assessing caspase activity can be used for other cell types, including both primary and transformed.
1. Differentiating bone marrow-derived macrophages (BMDMs)
NOTE: Perform all the steps in a tissue culture laminar flow hood, and use sterile aseptic techniques.
<ol>
	<li>Prepare a 1 mL syringe with a 21 G needle.</li>
	<li>Add 5 mL of HBSS + 2% FBS to a 15 mL tube.</li>
	<li>Using sterile forceps, take an excised femur, and insert the needle into the opening of the femur. Holding the femur in the HBSS + 2% FBS, flush the bone marrow out until the bone is white. Flush the tibia in the same manner. Continue to flush the solution to obtain a single-cell suspension.</li>
	<li>Centrifuge the single-cell suspension at 200 x g for 4 min at room temperature (RT).</li>
	<li>Remove the supernatant using a vacuum aspirator. Tap the tube to gently resuspend the pellet. Add 1 mL of red cell lysis buffer (see Table of Materials), and mix gently with a P1,000 pipette. Incubate at RT for 1 min.</li>
	<li>Add 10 mL of HBSS + 2% FBS, and centrifuge at 200 x g for 4 min at RT.</li>
	<li>Remove the supernatant, and resuspend in 10 mL of bone marrow-derived macrophage (BMDM) culture medium. 
	NOTE: BMDM culture medium consists of low-glucose DMEM with the addition of 10% FBS, 20 ng/ml M-CSF, penicillin (50 U/mL), and streptomycin (50 &#956;g/mL) (see Table of Materials). Alternatively, 20% L929 conditioned medium can be substituted for the M-CSF.</li>
	<li>Add 15 mL of BMDM culture medium into two 15 cm Petri dishes. Add 5 mL of the single-cell solution to each plate. 
	NOTE: Do not use tissue culture-treated plates. Tissue culture-treated plates limit the differentiation.</li>
	<li>Place the dishes at 37 &#176;C, 5% CO2, for 6 days. 
	&#8203;NOTE: The BMDMs can be harvested after 5-7 days. Longer incubation times for differentiation lead to increased anti-apoptotic protein expression26.</li>
</ol>
2. Harvesting, seeding, and treatment of cells
NOTE: Perform all the steps in a tissue culture laminar flow hood, and use sterile aseptic techniques. Fully differentiated macrophages adhere to the plate, allowing for easy separation, while floating cells can be discarded. Phosphate-buffered saline (PBS) can be used with or without Ca2+ and Mg2+.
<ol>
	<li>After 6 days of incubation, remove the floating cells and medium from the plate using an aspirator.</li>
	<li>Add 5 mL of PBS to each 15 cm plate. Remove the PBS from the plate using an aspirator.</li>
	<li>Add 2 mL of trypsin (see Table of Materials) to each plate. Incubate the plate until gentle tapping of the plate dislodges the cells. Take 5 mL of BMDM culture medium, and harvest the cells off the plate.
	<ol>
		<li>Transfer to the second 15 cm plate, and harvest the cells off the plate. Remove the cell suspension from the plate into a 50 mL tube. Take an additional 5 mL of BMDM culture medium, and wash both plates to ensure all the cells have been collected. Place the cell suspension into the same 50 mL tube.</li>
	</ol>
	</li>
	<li>Take 10 &#956;L of the cell suspension, and count using a hemacytometer using a 1:1 dilution with trypan blue. 
	NOTE: Automatic cell counters can be used as well when calibrated for the size of the macrophages.</li>
	<li>Seed the macrophages at a density of 1 x 106 cells/mL. BMDMs seeded at 2 x 106 cells/well in a 6-well plate provide approximately 2 mg/mL total protein.&#160;Allow the cells to adhere to the plate for a minimum of 6 h before treatment. 
	NOTE: For other cell types, the optimal concentration must be determined.</li>
	<li>Treat the macrophages with Smac mimetic (Compound A, see Table of Materials)22 at 250 nM and 500 nM for 16 h. 
	&#8203;NOTE: Include a positive control to induce apoptosis and caspase-3 cleavage to ensure the assay is working. Common apoptosis inducers include staurosporine and etoposide. For many cell lines to undergo apoptosis, 0.1-10 &#956;M for 16 h of stimulation is sufficient.</li>
</ol>
3. Preparing cell lysates from treated cells
NOTE: This step must be done on ice, and the reagents and materials must be pre-cooled.
<ol>
	<li>Transfer the plates containing the treated cells onto the ice. Collect the medium from the cell culture into a 1.5 mL tube, centrifuge at 300 x g for 5 min at 4 &#176;C, aspirate the medium, and put the tube on ice. This allows the collection of the cells that have detached from the plate.</li>
	<li>Add 1 mL of cold PBS to the cell culture plate for washing the cells, and aspirate all the PBS. Add 100 &#956;L of trypsin to the cells (if working with a 6-well dish). Allow the trypsin to lift the cells off the plate, and collect them into the 1.5 mL tube. Use 1 mL of cold PBS to ensure all the cells have been collected. 
	NOTE: If working with suspension cells, gently transfer the medium and cells to a 1.5 mL tube.</li>
	<li>Centrifuge the cells at 300 x g for 5 min at 4 &#176;C. Remove the supernatant, and resuspend in 100 &#956;L DISC lysis buffer (150 mM sodium chloride, 2 mM EDTA, 1% Triton X-100, 10% glycerol, 20 mM Tris, pH 7.5, see Table of Materials).</li>
	<li>Incubate the samples on ice for 20 min.</li>
	<li>Centrifuge the lysates at ~12,000 x g for 10 min at 4 &#176;C to pellet the insoluble fraction.</li>
	<li>Transfer 25 &#956;L of the lysate into a white flat-bottomed 96-well plate for the caspase-3/caspase-7 activity assay (step 5). 
	NOTE: Do not disturb the pellet. This is the insoluble fraction of the cell lysate.</li>
	<li>Transfer 10 &#956;L of the remaining lysate into a transparent flat-bottomed 96-well plate for the bicinchoninic acid (BCA) assay (step 4). This will be used for the normalization of the samples.</li>
	<li>Keep both plates on ice for further processing. 
	&#8203;NOTE: At this stage, the plates can be sealed with an adhesive cover and stored at &#8722;20 &#176;C for approximately 4 weeks.</li>
</ol>
4. Protein quantification using the BCA assay
NOTE: Other reagents or assays can be used to quantitate the amount of protein in each sample. In the population-based assay, the samples can be compared by normalizing the amount of protein used in the assay.
<ol>
	<li>Prepare standard protein concentrations between 0 &#956;g/mL and 2,000 &#956;g/mL (0 &#956;g/mL, 25 &#956;g/mL, 125 &#956;g/mL, 250 &#956;g/mL, 500 &#956;g/mL, 750 &#956;g/mL, 100 &#956;g/mL, 1,500 &#956;g/mL, 2,000 &#956;g/mL) with bovine serum albumin (BSA). Prepare blanks with lysis buffer only.</li>
	<li>Add 10 &#956;L of each standard into the flat-bottomed 96-well plate containing the samples, mentioned in step 3.7.</li>
	<li>Mix BCA reagent 1 with BCA reagent 2 in a 50:1 ratio (see Table of Materials). Add 200 &#956;L of mixed BCA reagent to each sample and standard.</li>
	<li>Incubate at 37 &#176;C for 30 min.</li>
	<li>Measure the absorbance at 562 nm on a fluorometric instrument, and quantify the protein concentration with the standard curve.</li>
</ol>
5. Population-based assay for caspase-3/caspase-7 activity
NOTE: Do not allow the cell lysates in the plate to sit on ice for more than 3 h. If caspase activity is present, this increases over time despite the sample being on ice. If the samples were frozen, thaw them on ice, and proceed immediately once the lysates have thawed.
<ol>
	<li>Start the fluorometric instrument (see Table of Materials) and heat the machine to 37 &#176;C. Prepare the script as mentioned:
	<ol>
		<li>Perform individual readings every minute for 40 min in order to determine the kinetics of the reaction.</li>
		<li>Set the excitation at 360 nm and the emission at 465 nm. Ten flashes per well are sufficient.</li>
	</ol>
	</li>
	<li>Prepare the positive control of recombinant caspase-3 (see Table of Materials). Mix 1 U of the recombinant caspase-3 enzyme in 50 &#956;L of lysis buffer (tube 1). Add 25 &#956;L of lysis buffer to an additional three tubes. Transfer 25 &#956;L from tube 1 to tube 2. Mix by pipetting.
	<ol>
		<li>Repeat for tube 3 and tube 4. Tube 5 will contain only 50 &#956;L of lysis buffer. Add 25 &#956;L of each standard into the white flat-bottomed 96-well plate for the caspase-3 activity assay, mentioned in step 3.6.</li>
	</ol>
	</li>
	<li>Prepare a master reaction mix for caspase activity assay on ice. For one reaction, mix 50 &#956;L of 2x caspase cleavage buffer (0.2M HEPES pH 7.5; 20% sucrose or PEG; 0.2% CHAPS), 5 &#956;L of 1 mM DEVD-AMC (caspase-3 tetrapeptide substrate), 2 &#956;L of 500 mM DTT, and 18 &#956;L of deionized water (see Table of Materials).</li>
	<li>Add 75 &#956;L of the reaction mix to each sample and standard to obtain a total reaction volume of 100 &#956;L.</li>
	<li>Immediately measure the fluorescence using the fluorometric instrument set up in step 5.1.</li>
	<li>For each fluorescent measurement, subtract the fluorescence reading for the blank (lysis buffer only) from the sample&#39;s fluorescence. Normalize the reading by dividing by the protein concentration of the sample (calculated in step 4.5)27: 
	<img alt="Equation 1" src="/files/ftp_upload/64745/64745eq01.jpg" style="margin: auto;" /></li>
	<li>Calculate the rate of caspase activity by determining the slope of the normalized fluorescence on the y-axis and time on the x-axis.</li>
</ol>
6. Single-cell assay (flow cytometry analysis) for caspase-3/caspase-7 activity
<ol>
	<li>Seed the cells as described in section 2.</li>
	<li>Harvest the cells into a 5 mL polystyrene tube. If working with adherent cells, collect the medium into the 5 mL tube. Add 1 mL of cold PBS to the cell culture plate, and collect the PBS into the 5 mL tube. Add trypsin to the cells (100 &#956;L if working with 6-well dishes). Allow the trypsin to lift the cells off the plate, and collect into the 5 mL tube. Use 1 mL of cold PBS to ensure all the cells have been collected. 
	NOTE: If working with suspension cells, gently transfer the medium and cells to a 5 mL tube.</li>
	<li>Centrifuge the samples at 300 x g for 5 min, 4 &#176;C. Remove the supernatant using a vacuum aspirator.</li>
	<li>Prepare the staining mix. The optimal staining concentration is 1 x 106-2 x 106 cells in 50 &#956;L. Dilute the fluorogenic substrate as per the manufacturer&#39;s instructions (see Table of Materials, flow cytometry). Take 1 &#956;L of the stock substrate, and dilute in 150 &#956;L of PBS. Add 50 uL per sample.</li>
	<li>Incubate the samples at 37 &#176;C for 30 min protected from light, with mixing every 15 min.</li>
	<li>With the flow cytometer utilized in this study, use the red laser at 640 nm, and detect using 675/25 nm. Run the unstained control sample first, and acquire a minimum of 10,000 events of the desired population. Exclude the debris using a gate (P1) on an FSC-A and SSC-A dot plot.
	<ol>
		<li>Use a histogram displaying the events in the P1 gate and detection for the caspase substrate (y-axis) to determine the median fluorescence intensity (MFI). 
		NOTE: The caspase substrate described in this method requires an excitation at 590 nm and emits at 628 nm.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to disclose.

In this method, a fluorogenic substrate is used in a population-based assay or single-cell analysis to measure caspase-3/caspase-7 activity. Both methods measure the caspase activity in a quantitative manner based on the cleavage of a substrate. One advantage is the ability to utilize these methods for numerous samples. With these methods, caspase-3/caspase-7 activity is detected in primary macrophages treated with Smac mimetics.
A critical aspect of the population-based fluorometric assay is the time from lysis to reading the fluorescence. The samples must be kept on ice throughout the procedure, particularly prior to "reading" the assay. This prevents premature cleavage and fluorescence of the substrate. Using the population-based assay, less optimization may be required. The amount of protein used in the assay is normalized, allowing the samples to be directly compared. One caveat is that at a late stage of apoptotic cell death, the total protein amount is reduced; hence, the detection of caspase activity may not be possible. Different kinetics or different treatment doses are recommended to circumvent this problem. In addition, other software can be used to accurately assess the rate of caspase activity besides the software described in this method. 
For the flow cytometric assay, enough events or cells are required to gate the populations confidently. In addition, more optimization in the flow-based assay may be required to achieve the optimal ratio of substrate to cell number. However, with flow cytometry, this method lends itself to measuring additional parameters, such as cell surface markers for cell type identification. 
Both the population and single-cell methods could be used for other caspases. However, it is important to remember that the recognition sequence is less discriminated for other caspases. As such, other methods for caspase activity must be used. This includes the inhibition of caspase activity, CRISPR or knock-down of specific caspases, and western blotting to detect the cleavage of known substrates. 
One alternative method for detecting caspase activity is time-lapse imaging. The same permeable caspase substrate could be used along with other markers of viability, such as annexin V, to provide information on the kinetics of cell death. Imaging would also separate the caspase activity and cell survival, allowing for the detection of sublethal amounts of caspase activity in a cell population. The non-lethal functions of caspase-3/caspase-7 are linked to antiviral regulation in innate immune cells29, particularly the activation of type I IFN via mitochondrial DNA release15,16. Thus, these assays to measure caspase activity are critical for identifying different modes of cell death and may be useful in assessing non-cell death functions.

Caspases are involved in several forms of programmed cell death. Apoptosis is the most studied form of programmed cell death and is associated with caspase activity1. All caspases possess a large and small catalytic subunit. Caspase-1, caspase-4, caspase-5, caspase-9, and caspase-11 possess a caspase activation and recruitment domain (CARD), and caspase-8 and caspase-10 contain death effector domains (DED)2,3,4,5 (Table 1). Apoptosis can be initiated by two major pathways: the extrinsic pathway and the intrinsic pathway. The extrinsic apoptotic pathway is triggered by death receptors, which are part of the tumor necrosis factor superfamily (TNFSF). Death receptors possess DED domains, facilitating caspase-8 activity6. The intrinsic apoptotic pathway involves the activation of caspase-9 after the formation of the apoptosome, requiring the release of cytochrome c and Apaf-17. The activation of either initiator caspase, caspase-8 or caspase-9, leads to the cleavage and subsequent activation of the executioner caspases, which are caspase-3, caspase-6, and caspase-7. Identifying that the executioner caspases are active indicates that the cells are undergoing apoptosis, and this activation is considered an important factor in defining the mode of cell death.
Caspase activation is also a critical juncture for regulating inflammation and the induction of alternative forms of programmed cell death. For example, caspase-1 activation leads to the maturation of pro-inflammatory cytokines of the interleukin-1 family8. The release and activation of cytokines from this family, particularly IL-1&#946; and IL-18, result from gasdermin D cleavage and pore formation at the plasma membrane9,10. Inadequate membrane repair of gasdermin D pores can result in a type of cell death known as pyroptosis11. Moreover, caspase-8 activity results in the inhibition of a caspase-independent cell death known as necroptosis12. Receptor-interacting serine/threonine protein kinase 1 (RIPK1) is one of the critical factors in necroptosis and in driving inflammation regulated by NF-kB. Models have shown that RIPK1 is cleaved by caspase-8, resulting in limiting NF-kB signaling, apoptosis, and necroptosis13,14. Therefore, identifying the activity of different caspases can aid in understanding the resulting inflammation and cell death modality.
Independent of the function of caspases in regulating cell death modalities, caspase activity can also regulate other cytokine families, such as interferon (IFN), in response to infection15,16. Additionally, caspases are involved in non-cell death functions, including cell fate decisions, tissue repair and regeneration, tumorigenesis through DNA repair, and neuronal synapse function. The activity of caspases in these non-lethal roles is thought to be limited by the cellular localization and amount of caspases. Therefore, quantifying the level of caspase activity may well define whether a cell undergoes cell death or whether the caspase plays a role in a non-cell death function4,17,18.
Caspase activity can be assessed by multiple methods. Western blotting for cleaved caspases and their substrates has been used as an indicator of activity, but these assays are qualitative at best. To determine whether caspase activity is associated with cell death, a quantitative measurement is ideal. Since caspases cleave substrates at a recognition site consisting of four amino acids, colorimetric, luminescence, or fluorometric methods have been developed. However, caspases appear to have plasticity in their substrate recognition19,20. The recognition sequence is not associated with the protein domains (Table 1). The tetrapeptide sequence DEVD, however, can be used to detect caspase-3 and caspase-7 activity20,21.
Smac mimetics are compounds targeting the inhibitors of apoptosis proteins (IAPs). The use of Smac mimetics in a subset of cancer cells causes the cells to become sensitive to TNF-induced cell death22. In primary macrophages, Smac mimetics cause cell death without the exogenous addition of TNF23,24. The loss of cIAP1 by Smac mimetic-induced degradation results in the production of TNF. If caspase activity is detected, this means the cells did not die by necroptosis but in an apoptotic manner. In this method, the detection of cleaved DEVD substrate is used to identify caspase-3/caspase-7 activity. Further experiments to confirm apoptotic cell death have been published previously24.

<table><tbody><tr><td>1.5 mL microfuge tubes</td><td>Sarstedt</td><td>72.706.400</td><td></td></tr><tr><td>15 cm Petri plates</td><td>Sarstedt</td><td>82.1184.500</td><td></td></tr><tr><td>37 degree incubator shaker</td><td>IKA shaker KS 4000i</td><td>97014-816</td><td>distributed by VWR</td></tr><tr><td>6-well cell culture dish</td><td>Sarstedt</td><td>83.392</td><td></td></tr><tr><td>96 well flat bottom, white polystyrene, non-sterile</td><td>Sigma</td><td>CLS3600</td><td></td></tr><tr><td>96 well flat plate</td><td>Sarstedt</td><td>82.1581</td><td></td></tr><tr><td>Ac-DEVD-AFC</td><td>Enzo Life Sciences</td><td>ALX-260-032-M005</td><td>Caspase-3 substrate</td></tr><tr><td>BD Fortessa</td><td>BD</td><td></td><td>any flow cytometer with the appropriate excitation and emission detector will work</td></tr><tr><td>b-glycerolphosphate</td><td>Sigma</td><td>G9422-10G</td><td></td></tr><tr><td>caspase-3 recombinant&amp;nbsp;</td><td>Enzo Life Sciences</td><td>ALX-201-059-U025</td><td></td></tr><tr><td>CHAPS</td><td>Sigma</td><td>1.11662</td><td></td></tr><tr><td>DMEM, low glucose, pyruvate</td><td>Thermoscientific</td><td>31885023</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418-250ML</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>03685-1KG</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>324626-25GM</td><td></td></tr><tr><td>Etoposide</td><td>MedChem Express</td><td>HY-13629</td><td></td></tr><tr><td>FBS</td><td>Thermoscientific</td><td>26140</td><td></td></tr><tr><td>Flow cytometry tubes</td><td>Falcon</td><td>352008</td><td></td></tr><tr><td>Flowjo</td><td>Flowjo</td><td></td><td>A license in required but any program that can analyze .fcs files will suffice</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516-500ML</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034</td><td></td></tr><tr><td>Magic Red caspase-3/7 assay kit; flow cytometry or imaging</td><td>Immunochemistry Technologies</td><td>935</td><td></td></tr><tr><td>M-CSF</td><td>ebioscience</td><td>14-8983-80</td><td>now a subsidiary of Thermoscientific</td></tr><tr><td>M-Plex</td><td>Tecan</td><td></td><td>any fluorometric reader will work with the appropriate excitation and emission detectors</td></tr><tr><td>NaCl</td><td>Roth</td><td>3957.1</td><td></td></tr><tr><td>PBS pH 7.4</td><td>Thermoscientific</td><td>10010023</td><td></td></tr><tr><td>Penicillin-Streptomycin-Glutamine (100X)</td><td>Thermoscientific</td><td>10378016</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>ThermoFisher Scientific</td><td>23225</td><td>protein concentration assay</td></tr><tr><td>protease inhibitors</td><td>Biomol</td><td>P9070.100</td><td></td></tr><tr><td>Smac mimetic, Compound A</td><td>Tetralogics</td><td></td><td>also known as 12911; structure shown in supplementary figures of Vince et al., Cell 2007</td></tr><tr><td>Sodium Fluoride</td><td>Sigma</td><td>S7920-100G</td><td></td></tr><tr><td>sodium orthovanadate</td><td>Sigma</td><td>S6508-10G</td><td></td></tr><tr><td>sodium pyrophosphate</td><td>Sigma</td><td>P8010-500G</td><td></td></tr><tr><td>Staurosporine</td><td>MedChem Express</td><td>HY-15141</td><td></td></tr><tr><td>sucrose</td><td>Sigma</td><td>1.07687</td><td></td></tr><tr><td>Tris Base</td><td>Sigma</td><td>T1503-1KG</td><td></td></tr><tr><td>Triton X100</td><td>Sigma</td><td>T8787-50ML</td><td></td></tr><tr><td>TrypLE</td><td>Thermoscientific</td><td>A1285901</td><td>In the protocol, it is listed as Trypsin</td></tr></tbody></table>

measuring caspase activity using a fluorometric assay or flow cytometry

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

Primary mouse macrophages were differentiated for 6 days. After 6 days, the cells were harvested, counted, and seeded. The following treatments were used: no treatment and Smac mimetic (Compound A)22 at 250 nM and 500 nM for 16 h (Figure 1). The experiment was performed in duplicate to allow caspase-3/caspase-7 activation to be assessed either by a population-based assay or single-cell analysis using flow cytometry.
The protein concentration of the cell lysates was quantified using the BCA assay (Supplementary Table 1). This is necessary to ensure the amount of protein used in the caspase-3/caspase-7 activity assay is the same between samples. In this population-based assay, the data can be presented in two ways. The first is to show the kinetics by plotting the adjusted fluorescence (y-axis) versus time (x-axis) (Figure 2A). Alternatively, the slope can be calculated to directly compare the samples (Figure 2B). The increase in slope upon 500 nM Smac mimetic treatment was not significant based on an ordinary one-way ANOVA with multiple comparisons (Dunnett&#39;s multiple comparison test28).
For the analysis of caspase-3/caspase-7 activity using flow cytometry, the cells and supernatant were harvested. Unstained cells or fluorescence minus one were used as a negative control, as well as the untreated cells. A histogram of the cells collected by flow cytometry showed a shift in fluorescence for the cells treated with Smac mimetics in comparison to the untreated cells (Figure 2C). The data can be shown either as median fluorescence intensity (Figure 2D) or as a fold change over the untreated cells (Figure 2E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64745/64745fig01.jpg" /> 
Figure 1: Flow chart of the study. (A) Femurs and tibias were excised from C57Bl/6 mice. The bones were flushed and differentiated in 20 ng/mL M-CSF for 6 days. (B) On day 6, macrophages were harvested and reseeded for treatment. One set of cells was harvested for lysates and assessed by fluorogenic activity, while the other set was harvested, incubated with the fluorogenic substrate, and assessed by flow cytometry. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64745/64745fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64745/64745fig02.jpg" /> 
Figure 2: Kinetic assay for caspase-3/caspase-7 activity and substrate cleavage by flow cytometry. (A,B) Representative data of the kinetic assay for caspase-3/caspase-7 activity (C-E) and substrate cleavage by flow cytometry. The macrophages were treated with two concentrations of Smac mimetic (Compound A; 250 nM and 500 nM) for 16 h. (A) Detection of the cleaved DEVD AFC substrate over time. The data were normalized to the protein concentration in the sample. (B) The rate of cleavage (slope) of DEVD AFC for each sample was normalized to the untreated slope and presented as the fold change over the untreated cells. (C) Flow cytometric histograms of the cells incubated with the caspase-3 substrate. (D,E) MFI comparison and fold change in the MFI compared to the untreated sample. Each data point represents an independent sample; mean &#177; standard error of the mean are shown; *p &#60; 0.05 using a one-way ANOVA and multiple comparison tests (Dunnett&#39;s multiple comparison test). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64745/64745fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Caspase</td>
			<td>Species&#160;</td>
			<td>Substrate sequence</td>
			<td colspan="3">Protein domains</td>
		</tr>
		<tr>
			<td>Caspase-1</td>
			<td>Hs, Mm</td>
			<td>(W/L)EHD</td>
			<td colspan="3">CARD, large domain, small catalytic domain</td>
		</tr>
		<tr>
			<td>Caspase-2</td>
			<td>Hs, Mm</td>
			<td>DEXD</td>
			<td colspan="3">CARD, large domain, small catalytic domain</td>
		</tr>
		<tr>
			<td>Caspase-4</td>
			<td>Hs</td>
			<td>(W/L)EHD</td>
			<td colspan="3">CARD, large domain, small catalytic domain</td>
		</tr>
		<tr>
			<td>Caspase-5</td>
			<td>Hs</td>
			<td>(W/L)EHD</td>
			<td colspan="3">CARD, large domain, small catalytic domain</td>
		</tr>
		<tr>
			<td>Caspase-9</td>
			<td>Hs, Mm</td>
			<td>(I/V/L)E(H/T)D</td>
			<td colspan="3">CARD, large domain, small catalytic domain</td>
		</tr>
		<tr>
			<td>Caspase-11</td>
			<td>Mm</td>
			<td>(W/L)EHD</td>
			<td colspan="3">CARD, large domain, small catalytic domain</td>
		</tr>
		<tr>
			<td>Caspase-12</td>
			<td>Mm</td>
			<td>ATAD</td>
			<td colspan="3">CARD, large domain, small catalytic domain</td>
		</tr>
		<tr>
			<td>Caspase-8</td>
			<td>Hs, Mm</td>
			<td>(I/V/L)E(H/T)D</td>
			<td colspan="3">DED, large domain, small catalytic domain</td>
		</tr>
		<tr>
			<td>Caspase-10</td>
			<td>Hs</td>
			<td>(I/V/L)E(H/T)D</td>
			<td colspan="3">DED, large domain, small catalytic domain</td>
		</tr>
		<tr>
			<td>Caspase-3</td>
			<td>Hs, Mm</td>
			<td>DEXD</td>
			<td colspan="3">large domain, small catalytic domain</td>
		</tr>
		<tr>
			<td>Caspase-6</td>
			<td>Hs, Mm</td>
			<td>(I/V/L)E(H/T)D</td>
			<td colspan="3">large domain, small catalytic domain</td>
		</tr>
		<tr>
			<td>Caspase-7</td>
			<td>Hs, Mm</td>
			<td>DEXD</td>
			<td colspan="3">large domain, small catalytic domain</td>
		</tr>
		<tr>
			<td>caspase-14</td>
			<td>hs, mm</td>
			<td>(W/L)EHD</td>
			<td colspan="3">large domain, small catalytic domain</td>
		</tr>
		<tr>
			<td>CARD</td>
			<td colspan="5">caspase activation and recruitment domain</td>
		</tr>
		<tr>
			<td>DED</td>
			<td colspan="5">death effector domain</td>
		</tr>
		<tr>
			<td>Hs</td>
			<td colspan="5">homo sapien</td>
		</tr>
		<tr>
			<td>Mm</td>
			<td colspan="5">mus musculus</td>
		</tr>
	</tbody>
</table>
Table 1: Substrate specificity and the protein domains of the caspases. The table is adapted from McStay et al.20; Shalini et al.3; and van Opdenbosch and Lamkanfi4.
Supplementary Table 1: DEVD kinetic analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64745/Supplementary Table 1. DEVD kinetic assay.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry at JoVE.com

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...

measuring-caspase-activity-using-fluorometric-assay-or-flow

Research

JoVE Journal

Biochemistry

4.2K Views.  University of Zürich. The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer. 

Video: Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Immunology and Infection

Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first to be activated in these pathways. This group includes caspase-2, -8, and -9, as well as the inflammatory caspases, caspase-1, -4, and -5. The initiator caspases are all activated by dimerization following recruitment to specific multiprotein complexes called activation platforms. Caspase Bimolecular Fluorescence Complementation (BiFC) is an imaging-based approach where split fluorescent proteins fused to initiator caspases are used to visualize the recruitment of initiator caspases to their activation platforms and the resulting induced proximity. This fluorescence provides a readout of one of the earliest steps required for initiator caspase activation. Using a number of different microscopy-based approaches, this technique can provide quantitative data on the efficiency of caspase activation on a population level as well as the kinetics of caspase activation and the size and number of caspase activating complexes on a per cell basis.

This protocol describes caspase Bimolecular Fluorescence Complementation (BiFC); an imaging-based method that can be used to visualize induced proximity of initiator caspases, which is the first step in their activation.

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first ...

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8, 16. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4, 14, 3, 11, 7.
In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15, 5, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15, 5, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.6, 10). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13, 12. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1, 10. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

Neuroscience

Flow Cytometric Analysis of Apoptotic Biomarkers in Actinomycin D-Treated SiHa Cervical Cancer Cells

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V and mitochondrial membrane potential) and late biomarkers (caspases 3 and 7, and DNA damage) of apoptosis were measured in experimental and control cultures. Cultures were incubated for 24 hours in a humidified incubator at 37 &#176;C with 5% CO2. The cells were then detached using trypsin and enumerated using a flow cytometric cell count assay. Cells were further analyzed for apoptosis using an Annexin V assay, a mitochondrial electrochemical transmembrane potential assay, a caspase 3/7 assay, and a DNA damage assay. This article provides an overview of apoptosis and traditional flow cytometry, and elaborates flow cytometric protocols for processing and analyzing SiHa cells. The results describe positive, negative, and sub-optimal experimental data. Also discussed are interpretation and caveats in performing flow cytometric analysis of apoptosis using this analytical platform. Flow cytometric analysis provides an accurate measurement of early and late biomarkers for apoptosis.

Apoptosis can be characterized by flow cytometric analysis of early and late apoptotic biomarkers. The cervical cancer cell line, SiHa, was analyzed for apoptosis biomarkers after treatment with Actinomycin D using a benchtop flow cytometer.

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V ...

Cancer Research

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

Caspase-3 Activity in the Rat Amygdala Measured by Spectrofluorometry After Myocardial Infarction

Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still unclear. Our laboratory has developed a rat model of post-MI syndrome that displays impaired cardiac functions, neuronal loss in the limbic system, cognitive deficits and behavioral signs of depression. At the neuronal level, caspase-3 activation mediates post-MI apoptosis in different limbic regions, such as the amygdala &#8211; peaking at 3 days post-MI. Cognitive and behavioral impairments appear 2-3 weeks post-MI and these correlate statistically with measures of caspase-3 activity. The protocol described here is used to induce MI, collect amygdala tissue and measure caspase-3 activity using spectrofluorometry. To induce MI, the descending coronary artery is occluded for 40 min. The protocol for evaluation of caspase-3 activation starts 3 days after MI: the rats are sacrificed and the amygdala isolated rapidly from the brain. Samples are quickly frozen in liquid nitrogen and kept at -80 &#176;C until actual analysis. The technique performed to assess caspase-3 activation is based on cleavage of a substrate (DEVD-AMC) by caspase-3, which releases a fluorogenic compound that can be measured by spectrofluorometry. The methodology is quantitative and reproducible but the equipment required is expensive and the procedure for quantifying the samples is time-consuming. This technique can be applied to other tissues, such as the heart and kidneys. DEVD-AMC can be replaced by other substrates to measure the activity of other caspases.

Myocardial infarction (MI) is not only followed by impaired cardiac function but also by apoptosis in the amygdala, a brain region involved in the behavioral consequences of MI. This protocol describes how to induce MI, collect amygdala tissue and measure caspase-3 activity, a marker of apoptosis, therein.

Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still ...

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1&#946; and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1&#946; release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...

Evaluation of Caspase Activation to Assess Innate Immune Cell Death

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is the initiation of innate immune programmed cell death (PCD) to eliminate infected or damaged cells and propagate immune responses. However, excess PCD is associated with inflammation and pathology. Therefore, understanding the activation and regulation of PCD is a central aspect of characterizing innate immune responses and identifying new therapeutic targets across the disease spectrum.
This protocol provides methods for characterizing innate immune PCD activation by monitoring caspases, a family of cysteine-dependent proteases that are often associated with diverse PCD pathways, including&#160;apoptosis,&#160;pyroptosis, necroptosis, and PANoptosis. Initial reports characterized caspase-2, caspase-8, caspase-9, and caspase-10 as initiator caspases and caspase-3, caspase-6, and caspase-7 as effector caspases in apoptosis, while later studies found the inflammatory caspases, caspase-1, caspase-4, caspase-5, and caspase-11, drive pyroptosis. It is now known that there is extensive crosstalk between the caspases and other innate immune and&#160;cell death molecules across the previously defined PCD pathways, identifying a key knowledge gap in the mechanistic understanding of innate immunity and PCD and&#160;leading to the characterization of PANoptosis. PANoptosis is a unique innate immune inflammatory PCD pathway regulated by PANoptosome complexes, which integrate components, including caspases,&#160;from other cell death pathways.
Here, methods for assessing the activation of caspases in response to various stimuli are provided. These methods allow for the characterization of PCD pathways both in vitro and in vivo, as activated caspases undergo proteolytic cleavage that can be visualized by western blotting using optimal antibodies and blotting conditions. A protocol and western blotting workflow have been established that allow for the assessment of the activation of multiple caspases from the same cellular population, providing a comprehensive characterization of the PCD processes. This method can be applied across research areas in development, homeostasis, infection, inflammation, and cancer to evaluate PCD pathways throughout cellular processes in health and disease.

This protocol describes a comprehensive method for assessing caspase activation (caspase-1, caspase-3, caspase-7, caspase-8, caspase-9, and caspase-11) in response to both in vitro and in vivo (in mice) models of infection, sterile insults, and cancer to determine the initiation of cell death pathways, such as pyroptosis, apoptosis, necroptosis, and PANoptosis.

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is ...

Visualization of Caspase Activation in Human Monocyte-Derived Macrophages

Source: Bol&#237;var, B. E., et. al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63162">Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages.</a> J. Vis. Exp. (2022).
This video demonstrates the activation of caspase proteins in human macrophages using bimolecular fluorescence complementation reporter proteins. The pro-domains of caspase-1 are fused with non-fluorescent fragments of Venus proteins, which are recruited to inflammasome complexes. The proximity of inflammasomes induces the refolding and fluorescence of Venus fragments, confirming caspase activation in the macrophages.

Source: Bol&#237;var, B. E., et. al., Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages. J. Vis. Exp. 
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JoVE Encyclopedia of Experiments

Immunology

Immune Response

Immune Modulation

Detection of Inflammasome Activation via Fluorescence Microscopy

Source: den Hartigh, A. B. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57463">Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages.</a> J. Vis. Exp.&#160;(2018)
This video demonstrates a method to detect NLRP3 inflammasome activation in macrophages using fluorescent reporter probes. The cells are treated with potassium ionophores and glycine to induce inflammasome assembly and caspase-1 activation. Fluorescent reporter probes visualize activated caspase-1, and cells are stained with a fluorescent dye for nuclei visualization.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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