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 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 apoptosis, 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 cell death molecules across the previously defined PCD pathways, identifying a key knowledge gap in the mechanistic understanding of innate immunity and PCD and leading to the characterization of PANoptosis. PANoptosis is a unique innate immune inflammatory PCD pathway regulated by PANoptosome complexes, which integrate components, including caspases, 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.
The innate immune system acts as the first line of defense during infection and in response to sterile stimuli, such as tissue injury and alterations in homeostasis. Innate immune sensors on the cell surface and in the cytoplasm respond to pathogen- or damage-associated molecular patterns (PAMPs or DAMPs, respectively) to trigger inflammatory signaling pathways and cellular responses. One of the key processes of the innate immune response is the induction of cell death to remove infected or damaged cells and drive further innate and adaptive immune responses. Programmed cell death (PCD) is a highly conserved process across species, highlighting its evolutionary importance as an innate immune mechanism.
There are several innate immune PCD pathways that can be activated in all cell types. Caspases are a key family of highly conserved, intracellular, cysteine-dependent proteases that are critical across many PCD pathways, including the traditionally non-inflammatory apoptosis pathway, as well as inflammatory PCD pathways such as pyroptosis, necroptosis, and PANoptosis1,2,3,4,5. There are 11 human and 10 murine caspases that are well defined, as well as pseudo-caspases that may be functional, and most are constitutively expressed as inactive monomeric or dimeric pro-caspases that require cleavage for activation6,7. Caspases also contain important domains for the recruitment and formation of multiprotein complexes. These include the caspase activation and recruitment domain (CARD), which can be found in caspase-1, caspase-2, caspase-4, caspase-5, caspase-9, and caspase-11, or the death effector domain (DED), which is found in caspase-8 and caspase-10. Through both their proteolytic activity and their ability to form multiprotein complexes, caspases are critical drivers of innate immune PCD.
The role of caspases in innate immune PCD was first identified in apoptosis, where the initiator caspases, caspase-2, caspase-8, caspase-9, and caspase-10, activate the executioner caspases, caspase-3, caspase-6, and caspase-7, to drive cell death8,9,10,11,12. Initiator caspases can be activated by diverse signaling cascades; the extrinsic pathway activates caspase-8 through extracellular ligand-induced death receptor signaling, and the intrinsic pathway activates caspase-9 through the disruption of mitochondrial integrity13. Activated initiator caspases cleave the linker separating the large and small catalytic subunits of executioner caspases to produce their active forms. The executioner caspases then cleave their substrates to disassemble the cell, resulting in DNA degradation, membrane blebbing, nuclear fragmentation, and the release of apoptotic bodies14,15. This process typically ends in a non-lytic and non-inflammatory form of cell death when coupled with the immediate clearance of the dying cells by efferocytosis16. However, defects in efferocytosis or a lack of phagocytic cells can lead to the accumulation of apoptotic cells, which then undergo lytic and inflammatory cell death17,18.
The inflammatory caspases, including caspase-1 (human and mouse), caspase-4 and caspase-5 (human), and caspase-11 (mouse), have been discovered to be activated during a form of inflammatory innate immune PCD (III-PCD) called pyroptosis. Caspase-1 activation is associated with the formation of inflammasomes, which are multiprotein complexes containing a cytosolic innate immune sensor, an adaptor molecule (apoptosis-associated speck-like protein containing a CARD [ASC]), and caspase-1. The formation of this complex allows caspase-1 to undergo proximity-mediated autoproteolysis to release its active form, which can cleave target substrates including the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 and the pore-forming molecule gasdermin D (GSDMD)19,20,21,22,23. Caspase-11, caspase-4, and caspase-5 can also activate GSDMD without the upstream formation of the inflammasome after sensing PAMPs such as lipopolysaccharide (LPS)19,20. These caspases undergo dimerization followed by oligomerization and self-cleavage for activation upon binding to cytosolic LPS, which leads to non-canonical inflammasome activation24,25,26 and caspase-1 activation in a cell-intrinsic manner to induce IL-1β and IL-18 maturation20. The maturation and release of these pro-inflammatory cytokines characterize these caspases as "inflammatory." Additionally, the apoptotic caspase-8 has been found to localize to the inflammasome, providing a link between apoptotic and pyroptotic processes. Studies have found that the apoptotic caspase-8 is also critical for regulating another form of PCD called necroptosis. The loss of caspase-8 results in spontaneous receptor-interacting serine-threonine kinase 3 (RIPK3)-mediated mixed lineage kinase domain-like pseudokinase (MLKL) activation to drive the III-PCD pathway of necroptosis27,28,29,30,31,32,33,34,35.
While caspases have historically been classified as "apoptotic" or "inflammatory" based on the type of cell death they initiate, growing evidence suggests there is extensive crosstalk between the innate immune PCD pathways through caspases3,4. For instance, the inflammatory caspase-1 from inflammasomes cleaves the apoptotic caspase-7 at its canonical activation site34. Caspase-1 activation can also lead to the cleavage of apoptotic substrates such as poly(ADP-ribose) polymerase 1 (PARP1)36. In cells lacking GSDMD, caspase-1 can also cleave caspase-337,38. Additionally, the canonically apoptotic caspase-3 can cleave gasdermin E (GSDME) to induce PCD17,18 and also processes GSDMD into an inactive form40. Furthermore, caspase-8 recruitment to the inflammasome complex has been observed39,40,41,42,43,44,45, and caspase-8 is a key regulator of canonical and noncanonical inflammasome activation39. There are also overlapping and redundant roles for caspase-8 and caspase-1 in many inflammatory conditions, and innate immune PCD characterized by the activation of pyroptotic, apoptotic, and necroptotic components occurs across the disease spectrum39,46,47,48,49,50.
Based on this crosstalk between inflammatory and apoptotic caspases, a key gap in the mechanistic understanding of innate immunity and PCD was identified, leading to the discovery of PANoptosis. PANoptosis is a unique form of III-PCD that is activated in response to pathogens, PAMPs, DAMPs, and alterations in homeostasis and is regulated by PANoptosomes – multifaceted macromolecular complexes that integrate components from other cell death pathways44,50,51,52,53,54,55. The totality of the biological effects in PANoptosis cannot be individually accounted for by pyroptosis, apoptosis, or necroptosis alone3,4,35,36,39,46,47,48, as PANoptosis is characterized by the activation of multiple caspases, including caspase-1, caspase-11, caspase-8, caspase-9, caspase-3, and/or caspase-7, depending on the context44,48,49,50,51,52,53,54,56,57,58,59,60,61,62. PANoptosis has been increasingly implicated in infectious and inflammatory diseases, as well as in cancers and cancer therapies3,4,35,36,39,44,46,47,48,49,50,51,52,53,54,56
,57,58,59,60,61,62,63,64,65,66.
Given the essential role of caspases across cell death pathways, including in apoptosis, pyroptosis, necroptosis, and PANoptosis, it is important to develop techniques to characterize their activation and understand the full complexity of the PCD pathways. The protocol here details a method to stimulate cells and measure the subsequent activation of caspases (Figure 1). This method leverages the proteolytic cleavage of caspases, which is generally required for their activation, as a means to study them. Through western blotting, the protein sizes can be determined, allowing for the clear visualization and differentiation of inactive pro-caspases and their activated, cleaved forms.
The major advantages of this protocol are 1) its ability to assess the activation of multiple caspases in parallel from a single population of endogenous cells to more accurately determine PCD activation and 2) the use of relatively simple lab techniques that do not require extensive training or expensive equipment. Previous protocols have used western blotting, fluorescent reporters, or antibody staining to monitor caspase activation in culture supernatants, cell and tissue lysates, whole cells via microscopy, and in vivo67,68,69,70,71, but these techniques generally only monitor one or two caspases in a sample. Furthermore, while synthetic peptide substrates containing caspase cleavage sites that fluoresce upon cleavage have been used to monitor caspase activation in cell or tissue lysates69, these substrates can often be cleaved by more than one caspase, making it difficult to determine the specific activation of individual caspases in this system. Additionally, the use of western blotting rather than the use of fluorescent reporters or other tag-based methods allows researchers to use endogenous cells rather than creating specific cell lines with reporter genes. There are multiple advantages to using endogenous cells, including the fact that many immortalized cell lines are deficient in key cell death molecules72,73, which could affect the results. Additionally, using endogenous cells allows for the evaluation of diverse cell types, such as macrophages, epithelial cells, and endothelial cells, rather than a single lineage. Western blotting is also a relatively simple and cost-effective technique that can be carried out in labs around the world without the need for large, expensive equipment or complicated setups.
This protocol is widely applicable across biology to understand both the cell death-dependent and cell death-independent functions of caspases, including their scaffolding roles and functions in other inflammatory signaling pathways74. Applying this method allows for a unified approach in the study of innate immune PCD pathways and inflammatory signaling across diseases and conditions, and this protocol can be used to identify critical regulatory processes and mechanistic connections that will inform the development of future therapeutic strategies.
The animal use and procedures were approved by the St. Jude Children's Research Hospital Committee on the Use and Care of Animals.
1. Preparing the solutions
2. Isolating bone marrow-derived macrophages
NOTE: For this protocol, 6-10-week-old wild-type mice with intact PCD pathways or mutant mice with the PCD regulators, effectors, or molecules of interest deleted or altered can be used.
3. Differentiating the BMDMs and plating for the experiments
4. Stimulating or infecting the cells
CAUTION: The agents included in this protocol are potentially pathogenic and should be handled with the appropriate precautions in a biosafety level 2 (BSL2) facility with approval from the relevant institutional and governmental authorities.
5. Collecting the combined supernatant and protein lysate to be used for caspase western blots
6. Collecting the protein lysate to be used for caspase western blots
7. Performing western blotting using the lysates collected from the BMDMs following the steps above or from tissue homogenates
NOTE: If using tissue, it can be homogenized by hand or through a power-driven tissue homogenizer. The protocol by Simpson76 provides a detailed description of tissue homogenization.
PANoptosis has been observed in response to numerous bacterial, viral, and fungal infections and other inflammatory stimuli, as well as in cancer cells44,48,49,50,51,52,53,54,56,57,58,60,61,62. In these cases, the activation of multiple caspases has been reported. Using the example stimulations in this protocol that are known to induce PANoptosis, namely IAV, HSV1, and F. novicida44,48,50,51,53,57, it is expected that cleavage may be observed as a surrogate for the activation of multiple caspases (Figure 2A-C). Additionally, LPS + ATP is included as a control; this combination is a canonical NLRP3 inflammasome trigger that is expected to induce caspase-1 activation. The activation of caspase-1 can be visualized in response to each of these triggers by the cleavage of the p45 pro-form to the p20 active form (Figure 2D). The p20 form can then cleave GSDMD and initiate cell death19.
Similarly, the activation of the apoptotic initiator caspase, caspase-8, is observed, as denoted by the presence of the p18 cleaved form (Figure 2A–D). The weak activation of caspase-9 can also be observed in some cases with these stimuli (Figure 2A–D). Downstream of these initiators, the effector caspases caspase-3 and caspase-7 are activated; this activation is observed as the formation of the p17/19 cleaved form of caspase-3 and the p20 cleaved form of caspase-7 (Figure 2A–D). To date, the activation of caspase-11 has not been widely evaluated in PANoptosis, but its activation has been observed in some contexts54. Caspase-11 remains an important inflammatory caspase, and its activation can be observed by the formation of its p26 cleaved form. While some low-level caspase-11 activation is seen, robust activation is not observed in response to IAV, HSV1, or F. novicida infection or LPS + ATP stimulation (Figure 2A–D).
Cells lacking upstream PANoptosis sensors do not undergo cell death in response to PANoptosis-inducing stimuli. For example, IAV infection induces the formation of the ZBP1-PANoptosome48,51,53,57, and Zbp1-/- cells are protected from cell death during IAV infection (Figure 3A). Similarly, HSV1 and F. novicida infections induce the formation of the AIM2-PANoptosome44, and Aim2-/- cells fail to undergo robust cell death in response to these infections (Figure 3B,C). In cells that are deficient in the upstream sensor necessary for PANoptosome formation, the activation of caspases is significantly reduced or even eliminated, as can be seen by the reduction in the intensity of the cleaved bands in the western blots (Figure 2A–C). Similarly, cells lacking upstream inflammasome sensors are protected from cell death in response to their respective stimuli, and Nlrp3-/- cells do not undergo cell death in response to LPS + ATP (Figure 3D). These cells also show reduced caspase activation (Figure 2D).
Figure 1: Overview of the experimental procedure. Bone marrow is isolated and differentiated into bone marrow-derived macrophages. These cells are then stimulated with a trigger that activates innate immune signaling and cell death, such as infection or an inflammatory stimulus. After the cells activate and begin to undergo PCD, the lysate is collected and analyzed by western blotting to monitor caspase cleavage to their active forms. Abbreviations: BMDMs = bone marrow-derived macrophages; DAMPs = damage-associated molecular patterns; III-PCD = inflammatory innate immune programmed cell death; PAMPs = pathogen-associated molecular patterns; PCD = programmed cell death. Please click here to view a larger version of this figure.
Figure 2: Activation of caspases in response to stimuli. Caspase activation from cell culture lysates can be assessed by evaluating the size of the products run on an electrophoretic gel. (A–D) Immunoblot analysis of pro- (P45) and activated (P20) CASP1, pro- (P43) and cleaved (P36 and P26) CASP11, pro- (P35) and cleaved (P17/P19) CASP3, pro- (P35) and cleaved (P20) CASP7, pro- (P55) and cleaved (P44 and P18) CASP8, and pro- (P49) and cleaved (P37) CASP9 in WT and Zbp1−/−, WT and Aim2−/−, or WT and Nlrp3−/− BMDMs after (A) IAV infection, (B) HSV1 infection, (C) Francisella novicida infection, or (D) LPS + ATP stimulation. Images are representative of three independent experiments. Abbreviations: BMDMs = bone marrow-derived macrophages; CASP1 = caspase-1; CASP3 = caspase-3; CASP7 = caspase-7; CASP8 = caspase-8; CASP9 = caspase-9; CASP11 = caspase-11; HSV1 = herpes simplex virus 1; IAV = influenza A virus; LPS = lipopolysaccharide; WT = wild-type. Please click here to view a larger version of this figure.
Figure 3: Inhibition of cell death when caspase activation does not occur. (A–D) Representative images of cell death in WT and Zbp1−/−, WT and Aim2−/−, or WT and Nlrp3−/− BMDMs after (A) IAV infection, (B) HSV1 infection, (C) Francisella novicida infection, or (D) LPS + ATP stimulation. The red mask denotes dead cells. Scale bars = 50 µm. Images are representative of three independent experiments. Abbreviations: BMDMs = bone marrow-derived macrophages; HSV1 = herpes simplex virus 1; IAV = influenza A virus; LPS = lipopolysaccharide; WT = wild-type. Please click here to view a larger version of this figure.
Monitoring caspase cleavage and activation provides one of the most comprehensive pictures of innate immune PCD activation as part of the innate immune response. The protocol described here demonstrates a strategy to monitor caspase activation in response to IAV, HSV1, and F. novicida infections and the sterile trigger LPS + ATP, but numerous other stimuli can induce PCD and could be used in this method, as has been shown in several publications44,48,49,50,51,52,53,54,56,57,58,59,60,61,62. This procedure can also be used to characterize the innate immune PCD in response to new triggers, where the mechanisms of PCD remain unknown. Using a combination of wild-type and genetically deficient cells and comparing the activation of different caspases between these populations can provide new insights into the sensors and regulators involved in PCD in response to a given stimulus.
Several technical points should be considered when performing this method. First, as this method involves infecting or otherwise stimulating cells to induce cell death, it is important to use sterile techniques for the isolation of the cells and for the subsequent stimulations. Contamination can significantly affect the results, leading to caspase activation even in unstimulated conditions. To test for this, it is best to always include an unstimulated control when collecting the samples and loading gels for the caspase blots. Additionally, when plating the cells, it is important to ensure that the cells are counted accurately and that clumping does not occur. If the cells are clumped, inconsistent numbers of cells will be deposited in the wells, and the MOI and the amount of protein per well will be affected. When performing the western blots, it is good practice to compare the banding pattern of the uncleaved, pro-form of the caspase, as well as to monitor the cleaved form to ensure that consistent amounts of protein were collected and loaded across the wells. However, when there is a high level of caspase activation in one sample, this can cause a distortion in the signal, leading to what appears to be a reduction in the pro-form in the other samples, as we show with caspase-8 in HSV1 and F. novicida infections in this example (Figure 2B,C). This likely occurs due to uneven antibody binding caused by the presence of too much protein in a particular spot on the membrane, or because of imaging artifacts where the substrate becomes oversaturated in a single sample. Adjusting the antibody dilutions or imaging settings can help overcome this issue. Additionally, when establishing an infection or stimulation for the first time, using a time course can be beneficial to identify when the activation of each caspase occurs. Without a time course, the data may be misleading, as the individual time point selected could be too early or too late, causing caspase activation to be missed and inappropriate conclusions to be made about its involvement. Similarly, testing different MOIs for infectious stimuli can also be beneficial to identify the specific conditions under which caspase(s) are activated.
The electrophoresis step also requires precision. When preparing to load the samples onto the gel, the tubes should always be centrifuged first, and only the supernatant should be loaded. Insoluble protein can interfere with the running of the gel and the subsequent analysis of the data. The combined supernatant and protein lysate should be used for the detection of caspase-1 rather than a pure cell lysate; this will greatly improve the sensitivity of detection. Caspase-3, caspase-7, and caspase-8 can also be detected well in the combined supernatant and protein lysate. However, for best results when detecting caspase-11 and caspase-9, the protein lysate collected in the RIPA buffer is sufficient. As the relative abundance of each caspase within the lysate can be low, as much sample as possible should be loaded onto the gel. This requires slow and steady pipetting to avoid overflowing into the neighboring wells. If poor separation is observed between the pro- and cleaved forms of the caspases, the gel run time can be extended, or a lower percentage of acrylamide can be used to optimize the separation.
Finally, the antibodies listed in this protocol have been optimized for use with mouse BMDMs, and several groups have rigorously tested them using genetically deficient cells as controls to confirm there are no non-specific bands at the molecular weights of interest. While other antibodies may also work, the ones listed here are optimal. Additionally, many of these antibodies also work well in tissue lysates, and other cell populations can also be collected and stimulated in vitro for analysis. Using cell lines or primary cells from humans or other species is also possible but will require an additional assessment of the antibodies for optimization. Human anti-caspase-1 (1:1,000) and human anti-caspase-8 (1:1,000; see Table of Materials) have been successfully used for such assessments, and the same anti-caspase-3, anti-cleaved caspase-3, anti-caspase-7, and anti-cleaved caspase-7 antibodies included in this protocol can be used for both mouse and human cells44,56,62.
The importance of understanding PCD pathways, including pyroptosis, apoptosis, necroptosis, and PANoptosis, as an integral component of the innate immune response is growing, as crosstalk between PCD pathways is increasingly being characterized, and cell death molecules are being identified as therapeutic targets across the disease spectrum3,4. Continuing to identify the innate immune sensors that induce PCD as well as III-PCD and characterize the timing and interplay of caspase activation will improve the ability to therapeutically modulate innate immune PCD pathways and improve patient outcomes.
The authors have nothing to disclose.
We thank members of the Kanneganti lab for their comments and suggestions, and we thank J. Gullett, PhD, for scientific editing support. Work in our lab is supported by National Institutes of Health (NIH) grants AI101935, AI124346, AI160179, AR056296, and CA253095 (to T.-D.K.) and by the American Lebanese Syrian Associated Charities (to T.-D.K.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
0.45 μm filter | Millipore | SCHVU05RE | |
10 mL syringe | BD Biosciences | 309604 | |
12% polyacrylamide gel with 10 wells | Bio-Rad | 4561043 | |
12-well plate | Corning | 07-200-82 | |
18 G needle | BD Biosciences | 305195 | |
25 G needle | BD Biosciences | 305122 | |
50 mL tube | Fisher Scientific | 50-809-218 | |
70 μm cell strainer | Corning | 431751 | |
150 mm tissue culture dishes | Corning | 430597 | |
182-cm2 tissue culture flask | Genesee Scientific | 25-211 | |
Accessory white trans tray | Cytiva | 29-0834-18 | |
Anti–caspase-1 antibody | AdipoGen | AG-20B-0042-C100 | |
Anti–caspase-11 antibody | Novus Biologicals | NB120-10454 | |
Anti–caspase-3 antibody | Cell Signaling Technology | 9662 | |
Anti–caspase-7 antibody | Cell Signaling Technology | 9492 | |
Anti–caspase-8 antibody | Cell Signaling Technology | 4927 | |
Anti–caspase-9 antibody | Cell Signaling Technology | 9504 | |
Anti–cleaved caspase-3 antibody | Cell Signaling Technology | 9661 | |
Anti–cleaved caspase-7 antibody | Cell Signaling Technology | 9491 | |
Anti–cleaved caspase-8 antibody | Cell Signaling Technology | 8592 | |
Anti-mouse HRP-conjugated secondary antibody | Jackson ImmunoResearch Laboratories | 315-035-047 | |
Anti-rabbit HRP-conjugated secondary antibody | Jackson ImmunoResearch Laboratories | 111-035-047 | |
Anti-rat HRP-conjugated secondary antibody | Jackson ImmunoResearch Laboratories | 112-035-003 | |
Anti–β-Actin antibody (C4) HRP | Santa Cruz | sc-47778 HRP | |
ATP | InvivoGen | tlrl-atpl | |
BBL Trypticase Soy Broth | BD Biosciences | 211768 | |
Bead bath | Chemglass Life Sciences | CLS-4598-009 | |
Biophotometer D30 | Eppendorf | 6133000010 | |
BME | Sigma | M6250 | |
Bromophenol blue | Sigma | BO126 | |
Cell scrapers | CellTreat Scientific Products | 229315 | |
Chemiluminescence imager (Amersham 600) | Cytiva | 29083461 | |
CO2 chamber | VetEquip | 901703 | |
Cuvettes | Fisher Scientific | 14-955-129 | |
Dissecting scissors | Thermo Fisher Scientific | 221S | |
DMEM | Thermo Fisher Scientific | 11995-073 | |
DTT | Sigma | 43815 | |
Eelectrophoresis apparatus | Bio-Rad | 1658004 | |
Ethanol | Pharmco | 111000200 | |
Fetal bovine serum | Biowest | S1620 | |
Filter paper | Bio-Rad | 1703965 | |
Forceps | Fisher Scientific | 22-327379 | |
Francisella novicida (U112 strain) | BEI Resources | NR-13 | |
Gel releaser | Bio-Rad | 1653320 | |
Gentamycin | Gibco | 15750060 | |
Glycerol | Sigma | G7893 | |
Glycine | Sigma | G8898 | |
HCl | Sigma | H9892 | |
Heat block | Fisher Scientific | 23-043-160 | |
Herpes simplex virus 1 (HF strain) | ATCC | VR-260 | |
High glucose DMEM | Sigma | D6171 | |
Human anti–caspase-1 antibody | R&D Systems | MAB6215 | |
Human anti–caspase-8 antibody | Enzo | ALX-804-242 | |
Humidified incubator | Thermo Fisher Scientific | 51026282 | |
Image analysis software | ImageJ | v1.53a | |
IMDM | Thermo Fisher Scientific | 12440-053 | |
Influenza A virus (A/Puerto Rico/8/34, H1N1 [PR8]) | constructed per Hoffmann et al. | ||
L929 cells | ATCC | CCL-1 | cell line for creating L929-conditioned media |
L-cysteine | Thermo Fisher Scientific | BP376-100 | |
Luminata Forte Western HRP substrate | Millipore | WBLUF0500 | standard-sensitivity HRP substrate |
MDCK cells | ATCC | CCL-34 | cell line for determining IAV viral titer |
Methanol | Sigma | 322415 | |
Microcentrifuge | Thermo Fisher Scientific | 75002401 | |
Non-essential amino acids | Gibco | 11140050 | |
Nonfat dried milk powder | Kroger | ||
NP-40 solution | Sigma | 492016 | |
PBS | Thermo Fisher Scientific | 10010023 | |
Penicillin and streptomycin | Sigma | P4333 | |
Petri dish | Fisher Scientific | 07-202-011 | |
PhosSTOP | Roche | PHOSS-RO | |
Power source | Bio-Rad | 164-5052 | |
Protease inhibitor tablet | Sigma | S8820 | |
PVDF membrane | Millipore | IPVH00010 | |
Rocking shaker | Labnet | S2035-E | |
SDS | Sigma | L3771 | |
Sodium chloride | Sigma | S9888 | |
Sodium deoxycholate | Sigma | 30970 | |
Sodium hydroxide | Sigma | 72068 | |
Sodium pyruvate | Gibco | 11360-070 | |
Square Petri dish | Fisher Scientific | FB0875711A | |
Stripping buffer | Thermo Fisher Scientific | 21059 | |
Super Signal Femto HRP substrate | Thermo Fisher Scientific | 34580 | high-sensitivity HRP substrate |
Tabletop centrifuge | Thermo Fisher Scientific | 75004524 | |
Trans-Blot semi-dry system | Bio-Rad | 170-3940 | |
Tris | Sigma | TRIS-RO | |
Tween 20 | Sigma | P1379 | |
Ultrapure lipopolysaccharide (LPS) from E. coli 0111:B4 | InvivoGen | tlrl-3pelps | |
Vero cells | ATCC | CCL-81 | cell line for determining HSV1 viral titer |