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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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/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/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/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

4.1K 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

A Fluorescence-based Assay of Phospholipid Scramblase Activity

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and biochemically verified was opsin, the apoprotein of the photoreceptor rhodopsin. Rhodopsin is a G protein-coupled receptor localized in rod photoreceptor disc membranes of the retina where it is responsible for the perception of light. Rhodopsin&#39;s scramblase activity does not depend on its ligand 11-cis-retinal, i.e., the apoprotein opsin is also active as a scramblase. Although constitutive and regulated phospholipid scrambling play an important role in cell physiology, only a few phospholipid scramblases have been identified so far besides opsin. Here we describe a fluorescence-based assay of opsin&#39;s scramblase activity. Opsin is reconstituted into large unilamellar liposomes composed of phosphatidylcholine, phosphatidylglycerol and a trace quantity of fluorescent NBD-labeled PC (1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazole-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine). Scramblase activity is determined by measuring the extent to which NBD-PC molecules located in the inner leaflet of the vesicle are able to access the outer leaflet where their fluorescence is chemically eliminated by a reducing agent that cannot cross the membrane. The methods we describe have general applicability and can be used to identify and characterize scramblase activities of other membrane proteins.

We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin.

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and ...

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their involvement in two-component signal transduction, a ubiquitous system through which bacteria sense and respond to environmental stimuli. Two-component signaling features autophosphorylation of a histidine kinase, followed by phosphotransfer to the receiver domain of a response regulator protein, which ultimately leads to an output response. Autophosphorylation of the histidine kinase is responsive to the presence of a cognate environmental stimulus, thereby giving bacteria a means to detect and respond to changes in the environment. Despite their importance in bacterial biology, histidine kinases remain poorly understood due to the inherent lability of phosphohistidine. Conventional methods for studying these proteins, such as SDS-PAGE autoradiography, have significant shortcomings. We have developed a nitrocellulose binding assay that can be used to characterize histidine kinases. The protocol for this assay is simple and easy to execute. Our method is higher throughput, less time-consuming, and offers a greater dynamic range than SDS-PAGE autoradiography.

We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

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 ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often viruses exhibit cell and tissue tropism and are adapted to specific cell or tissue proteases. Moreover, these viruses can introduce mutations or insertions into their genome during replication that may affect the cleavage, and thus can contribute to adaptations to a new host. Here, we present a fluorogenic peptide cleavage assay that allows a rapid screening of peptides mimicking the cleavage site of viral fusion proteins. The technique is very flexible and can be used to investigate the proteolytic activity of a single protease on many different substrates, and in addition, it also allows exploration of the activity of multiple proteases on one or more peptide substrates. In this study, we used peptides mimicking the cleavage site motifs of the coronavirus spike protein. We tested human and camel derived Middle East Respiratory Syndrome coronaviruses (MERS-CoV) to demonstrate that single and double substitutions in the cleavage site can alter the activity of furin and dramatically change cleavage efficiency. We also used this method in combination with bioinformatics to test furin cleavage activity of feline coronavirus spike proteins from different serotypes and strains. This peptide-based method is less labor- and time intensive than conventional methods used for the analysis of proteolytic activity for viruses, and results can be obtained within a single day.

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most &#947;-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.

Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry.

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...