We thank the members of LBH&#39;s lab past and present who contributed to the development of this technique. This lab is supported by NIH/NIDDK T32DK060445 (BEB), NIH/NIDDK F32DK121479 (BEB), NIH/NIGMS R01GM121389 (LBH). Figure 2 was drawn using Biorender software.

This protocol follows the guidelines of Baylor College of Medicine&#39;s human research ethics committee for the manipulation of human samples. Blood samples are handled following the institutional safety guidelines for human samples. Blood samples are obtained at a regional blood bank, where they are collected with citrate phosphate dextrose (CPD) solution. However, blood collected with other anticoagulants like sodium heparin, lithium heparin, or EDTA can also be used for this protocol28,</...

<ol>
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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noncanonical+Roles+of+Caspase-4+and+Caspase-5+in+Heme-Driven+IL-1&#946;+Release+and+Cell+Death.">Noncanonical Roles of Caspase-4 and Caspase-5 in Heme-Driven IL-1&#946; Release and Cell Death.</a> The Journal of Immunology. 206 (8), 1878-1889 (2021).</li><li>Schmid-Burgk, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase-4+mediates+noncanonical+activation+of+the+NLRP3+inflammasome+in+human+myeloid+cells.">Caspase-4 mediates noncanonical activation of the NLRP3 inflammasome in human myeloid cells.</a> European Journal of Immunology. 45 (10), 2911-2917 (2015).</li><li>Baker, P. 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This protocol describes the workflow to obtain macrophages from monocytes isolated from human blood samples and a method to efficiently introduce the inflammatory caspase BiFC reporters into human MDM without compromising cell viability and behavior.
This protocol takes advantage of the BiFC technique35 to tag the inflammatory caspases at the caspase recruitment domain (CARD) with non-fluorescent fragments of the split fluorescent protein Venus. Inflammatory caspases en...

The caspases are a family of cysteine aspartate proteases that can be grouped into initiator caspases and executioner caspases. Executioner caspases comprise caspase-3, -6 and -7. They are naturally found in cells as dimers and are cleaved by the initiator caspases to execute apoptosis1. Initiator caspases include human caspase-1, -2, -4, -5, -8, -9, -10 and -12. They are found as inactive zymogens (pro-caspases) that are activated by proximity-induced dimerization and stabilized by auto-proteolytic cleavage2,3. The inflammatory caspases are a subset of the initiator caspases2 and encompass caspase-1, -4, -5, and -12 in humans, and caspase-1, -11, and -12 in mouse4,5. Rather than an apoptotic role, they play a central role in inflammation. They mediate proteolytic processing and secretion of pro-interleukin (IL)-1&#946; and pro-IL-186,7, which are the first cytokines to be released in response to pathogenic invaders8,9. Caspase-1 is activated upon recruitment to its activation platform; a large molecular weight protein complex termed the inflammasome (Figure 1A)10. Dimerization of caspase-4, -5, and -11 occurs independently of these platforms through a noncanonical inflammasome pathway11,12.
Canonical inflammasomes are cytosolic multimeric protein complexes that consist of an inflammasome sensor protein, the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), and the effector protein caspase-110. The most well studied canonical inflammasomes are the NOD-like receptor family containing a pyrin domain (NLRP), NLRP1 and NLRP3, the NLR family containing a CARD (NLRC), NLRC4, and the absent in melanoma 2 (AIM2). They each contain a pyrin domain, a CARD, or both domains. The CARD domain mediates the interaction between CARD-containing caspases and their upstream activators. Therefore, the scaffold molecule ASC, which is composed of an N-terminal pyrin domain (PYD) and a C-terminal CARD motif13,14, is required for recruitment of caspase-1 to the NLRP110, NLRP315, and AIM216 inflammasomes.
Each inflammasome is named after its unique sensor protein that recognizes distinct pro-inflammatory stimuli (Figure 1B). Activators of this pathway are termed canonical stimuli. Inflammasomes serve as sensors for microbial components and tissue stress, and assemble to trigger a robust inflammatory response through activation of the inflammatory caspases17. Inflammasome assembly initiates caspase-1 activation to mediate maturation and secretion of its main substrates pro-IL-1&#946; and pro-IL-18. This process occurs via a two-step mechanism. First, a priming stimulus upregulates the expression of certain inflammasome proteins and pro-IL-1&#946; through activation of the NF-&#954;B pathway. Second, an intracellular (canonical) stimulus induces inflammasome assembly and recruitment of procaspase-16,7.
Caspase-4 and caspase-5 are the human orthologs of murine caspase-1111. They are activated in an inflammasome-independent manner by intracellular lipopolysaccharide (LPS), a molecule found in the outer membrane of Gram-negative bacteria18,19,20, and by extracellular heme, a product of red blood cell hemolysis21. It has been proposed that LPS binds directly to the CARD motif of these proteins and induces their oligomerization20. Activation of caspase-4 or caspase-5 promotes IL-1&#946; release by inducing an inflammatory form of cell death called pyroptosis through cleavage of the pore-forming protein gasdermin D (GSDMD)18,19. In addition, the efflux of potassium ions resulting from caspase-4 and GSDMD-mediated pyroptotic death induces activation of the NLRP3 inflammasome and subsequent activation of caspase-122,23. Therefore, caspase-4, -5, and -11 are considered intracellular sensors for LPS that are able to induce pyroptosis and caspase-1 activation in response to specific stimuli11,24.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63162/63162fig01.jpg" /> 
Figure 1: Inflammatory caspases and caspase-bimolecular fluorescence complementation (BiFC) assay. (A) Diagram showing the caspase-BiFC system, where two caspase-1 prodomains (C1-pro) linked to each non-fluorescent fragment of Venus (Venus-C or Venus-N) are recruited to the NLRP3 activation platform, forcing Venus to refold and fluoresce. This complex appears as a green spot under the microscope and serves as a readout for inflammatory caspase-induced proximity, which is the first step in initiator caspase activation. (B)&#160;Schematic showing the domain organization of inflammasome components and inflammatory caspases. <a href="https://www.jove.com/files/ftp_upload/63162/63162fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Measuring specific initiator caspases activation is difficult, and there are not many methods available to do so by imaging approaches. Caspase Bimolecular Fluorescence Complementation (BiFC) can be used to visualize inflammatory caspase activation directly in living cells (Figure 1A)25. This technique has been recently adapted for use in human monocyte-derived macrophages (MDM)21. Caspase BiFC measures the first step in inflammatory caspase activation, induced proximity to facilitate dimerization. Expression of plasmids encoding the CARD-containing caspase prodomain fused to non-fluorescent fragments of the photostable yellow fluorescent protein Venus (Venus-C [VC]) and Venus-N [VN]) are used. When the two caspase prodomains are recruited to their activation platform or undergo induced proximity, the two halves of Venus are brought in close proximity and forced to refold and fluoresce (see Figure 1A,B). This provides a real-time readout of specific inflammatory caspase activation.
Human MDM abundantly express inflammasome genes and pattern recognition receptors that identify danger signals and pathogen products. This provides an ideal cell type for the interrogation of inflammatory caspase pathways. In addition, they can be derived from peripheral blood and even from patient samples to assess inflammatory caspase activation in a specific disease state. This protocol describes how to introduce the BiFC caspase reporters into MDM using nucleofection, an electroporation-based transfection method, how to treat the cells to induce inflammatory caspase activation, and how to visualize the active caspase complexes using microscopy approaches. Additionally, this methodology can be adapted to determine the molecular composition of these complexes, subcellular localization, kinetics, and size of these highly ordered structures25,26,27.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>48 well tissue culture2:34 plates</td>
			<td>Genesee Scientific</td>
			<td>25-108</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Tissue Culture Dishes</td>
			<td>VWR</td>
			<td>25382-166</td>
			<td></td>
		</tr>
		<tr>
			<td>2 Mercaptoethanol 1000x</td>
			<td>Thermo Fisher Scientific</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>8 well chambered coverglass with 1.5 HP coverglass</td>
			<td>Cellvis</td>
			<td>c8-1.5H-N</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoMACS columns</td>
			<td>Miltenyi (Biotec)</td>
			<td>130-021-101</td>
			<td>For automated separation using AutoMACS Pro Separator only</td>
		</tr>
		<tr>
			<td>AutoMACS Pro Separator</td>
			<td>Miltenyi (Biotec)</td>
			<td>130-092-545</td>
			<td>For automated separation using AutoMACS Pro Separator only</td>
		</tr>
		<tr>
			<td>AutoMACS Pro Washing Solution</td>
			<td>Miltenyi (Biotec)</td>
			<td>130-092-987</td>
			<td>For automated separation using AutoMACS Pro Separator only</td>
		</tr>
		<tr>
			<td>AutoMACS Rinsing Solution</td>
			<td>Miltenyi (Biotec)</td>
			<td>130-091-222</td>
			<td>For automated separation using AutoMACS Pro Separator only</td>
		</tr>
		<tr>
			<td>AutoMacs running buffer</td>
			<td>Miltenyi (Biotec)</td>
			<td>130-091-221</td>
			<td>For manual or automated separation using QuadroMACS or AutoMACS pro Separator</td>
		</tr>
		<tr>
			<td>Axio Observer Z1 motorized inverted microscope equipped with a CSU-X1A 5000 spinning disk unit</td>
			<td>Zeiss</td>
			<td></td>
			<td>Any confocal microscope equipped with a laser module fitted with laser lines of 568 nm (RFP) and 488 or 512 nm (GFP or YFP) wavelengths can be used</td>
		</tr>
		<tr>
			<td>AxioObserver A1, Research Grade Inverted Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Any epifluorescence microscope with fluorescence filters capable of exciting 568 nm (RFP) and 488 or 512 nm (GFP or YFP) wavelengths can be used</td>
		</tr>
		<tr>
			<td>CD14+ MICROBEADS</td>
			<td>Miltenyi (Biotec)</td>
			<td>130-050-201</td>
			<td>For manual or automated separation using QuadroMACS or AutoMACS pro Separator</td>
		</tr>
		<tr>
			<td>DPBS without calcium chloride and magnesium chloride</td>
			<td>Sigma</td>
			<td>D8537-6x500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>DsRed mito plasmid</td>
			<td>Clontech</td>
			<td>632421</td>
			<td>Similar plasmids that can be used as fluorescent reporters can be found on Addgene</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS 6 x 100 mL</td>
			<td>Sigma</td>
			<td>GE17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement (100x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050079</td>
			<td></td>
		</tr>
		<tr>
			<td>GM-CSF</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHC2011</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemin BioXtra, from Porcine, &#8805;96.0% (HPLC)</td>
			<td>Sigma</td>
			<td>51280-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630106</td>
			<td></td>
		</tr>
		<tr>
			<td>Inflammatory caspase BiFC plasmids</td>
			<td></td>
			<td></td>
			<td>Available by request from LBH lab</td>
		</tr>
		<tr>
			<td>LPS-EB Ultrapure</td>
			<td>Invivogen</td>
			<td>TLRL-3PELPS</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Miltenyi (Biotec)</td>
			<td>130-042-401</td>
			<td>For manual separation using QuadroMACS Separator only</td>
		</tr>
		<tr>
			<td>MACS 15 mL Tube Rack</td>
			<td>Miltenyi (Biotec)</td>
			<td>130-091-052</td>
			<td>For manual separation using QuadroMACS Separator only</td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi (Biotec)</td>
			<td>130-042-303</td>
			<td>For manual separation using QuadroMACS Separator only</td>
		</tr>
		<tr>
			<td>mCherry plasmid</td>
			<td>Yungpeng Wang Lab</td>
			<td></td>
			<td>Similar plasmids that can be used as fluorescent reporters can be found on Addgene</td>
		</tr>
		<tr>
			<td>Neon Transfection System</td>
			<td>Thermo Fisher Scientific</td>
			<td>MPK5000</td>
			<td>Includes Neon electroporation device, pipette and pipette station</td>
		</tr>
		<tr>
			<td>Neon Transfection System 10 &#181;L Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>MPK1096</td>
			<td>Includes resuspension buffer R, resuspension buffer T, electrolytic buffer E, 96 x 10 &#181;L Neon tips and Neon electroporation tubes</td>
		</tr>
		<tr>
			<td>Nigericin sodium salt, ready made solution</td>
			<td>Sigma</td>
			<td>SML1779-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine Hydrobromide</td>
			<td>Sigma</td>
			<td>P7280-5mg</td>
			<td></td>
		</tr>
		<tr>
			<td>QuadroMACS Separator</td>
			<td>Miltenyi (Biotec)</td>
			<td>130-090-976</td>
			<td>For manual separation using QuadroMACS Separator only</td>
		</tr>
		<tr>
			<td>qVD-OPh</td>
			<td>Fisher (ApexBio)</td>
			<td>50-101-3172</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875119</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 0.5 M EDTA, pH 8.0</td>
			<td>Thermo Fisher Scientific</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Zen 2.6 (blue edition) software</td>
			<td>Zeiss</td>
			<td></td>
			<td>Any software used to operate the confocal microscope of choice</td>
		</tr>
	</tbody>
</table>

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.

The scheme shown in Figure 2 gives an overview of how to obtain, transfect, and image human MDM. After incubation of the selected CD14+ monocytes with GM-CSF for 7 days, the cell morphology changes over the course of the differentiation period (Figure 3A), going from spherical suspension cells to spindly and fully attached (days 3 and 4), and lastly to more spread cells when fully differentiated (day 7). Fully differentiated cells are then detached from the plat...

Watch this Scientific Journal Video about Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages at JoVE.com

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

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 ...

This protocol is significant because it can be used to visualize and interrogate inflammatory caspase pathway at the molecular level in living cells in a particular disease state. The main advantage of this technique is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complexes in primary immune cells. With minor optimizations, this method can be adapted to other primary cells and a range of proteins activated by oligomerization, and its applicability is not limited to microscopic methodologies.First, aspirate the media from fully differentiated macrophages on 10 centimeter dishes and wash the cell monolayer with warm serum free RPMI-1640 medium, removing all the media completely. Harvest the cells by adding two milliliters of 0.25%warm trypsin-EDTA solution per 10 centimeter dish. Gently pipette the trypsin-EDTA up and down over the entire dish with a P-1000 micropipette, then transfer the suspension to a 15 milliliter conical tube containing five milliliters of warm complete culture medium.Check for cell detachment under a bright field microscope and detach again if needed. Aspirate the medium and resuspend the cells in 10 milliliters of 1X sterile PBS pre-warmed to 37 degrees Celsius. Then take a 20 microliter aliquot to determine the cell number using a hemocytometer.Take one to two times 10 to the fifth cells per transfection and place them in a 15 milliliter tube. Bring to a final volume of 15 milliliters with pre-warmed 1X sterile PBS. Aspirate the PBS and centrifuge one more time for one minute at 250 x g.Remove any residual PBS from the cell pellet using a P-200 micropipette. Take a 1.5 milliliter sterile microtube per transfection and add the appropriate reporter plasmid and caspase BiFC plasmids in the hood. Place the pipette station, device, tips, electroporation tubes, and pipette in a sterile laminar flow hood.Connect and switch on the nucleofection device. Enter the transection parameters in the startup screen displayed. Press on Voltage, enter 1000, and press on Done to set it to 1000 volts.Press on Width, enter 40, and press on Done to set the pulse to 40 milliseconds. Lastly, press on Pulses, enter 2, and press on Done to set the number of electrical pulses to 2. Take one of the electroporation tubes and fill it with three milliliter of electrolytic buffer E at room temperature.Insert the electroporation tube into the pipette holder on the pipette station, ensuring that the electrode on the side of the tube is facing inwards and a click sound is heard when the tube is inserted. Take the cell pellet and add 10 microliters of pre-warmed resuspension R buffer for each one to two times 10 to the fifth cells, and mix gently with a P-20 micropipette. Add 10 microliters of the cell suspension to each transfection tube and mix gently with a P-20 micropipette.Insert a tip into the pipette by pressing the push button to the second stop, ensuring that the clamp completely picks up the mounting stem of the piston in the tip. Next, press the push button on the pipette to the first stop and dip into the first tube containing cell or plasmid DNA mixture to aspirate the sample. Insert the pipette with the sample very carefully into the pipette holder, ensuring that the pipette clicks and is appropriately placed.Press start on the touch screen and wait until the electric pulses are delivered. Slowly remove the pipette from the station and immediately add the transected cell suspension into the corresponding well with the pre-warmed antibiotic-free medium by slowly pressing the push button to the first stop. Gently rock the plate with transected cells and incubate for one to three hours in a humidified tissue culture incubator, then add 200 microliters of pre-warmed culture medium to each well.Place the dish in the incubator and allow at least 24 hours for gene expression. The next day, inspect the cell viability and transfection efficiency using an epifluorescence microscope. Remove the media from the cells carefully with a P-1000 micropipette and add 500 microliters of the stimulus solution down the side of the well.To run untreated control wells, add an imaging medium without the stimulus. To visualize the cells using an epifluorescence or confocal microscope, turn on the microscope and the fluorescent light source following the instructions. Select the 10 times or 20 times objective and place the culture dish on the microscope stage.Find cells under the 568 nanometer filter with the eye piece. And focus on the cells expressing the reporter red cells. Count all the red cells in the visual field.While in the same visual field, change to the 488 or 512 filters, count the number red cells that are also green. Count at least 100 red cells of minimum three fields. Calculate the percentage of Venus-positive transected cells per visual field and average the resulting percentages for each treatment well to get the standard deviation.To image the cells using an epifluorescence or confocal microscope, visualize the live image of the cells on the computer screen as acquired by the camera. Fine tune the focus and position of the cells using the joystick control and focus wheel. Set the percentage laser power and exposure time for the 512 nanometer or 488 nanometer and 568 nanometer lasers, so that the signal in the image looks good without saturation.Turn on the live capture and examine the resulting image, ensuring that a distinct peak is seen for each floor in the display histograms for both channels. While visualizing the live image of the cells, take multiple representative images of a field that contains one or more cells expressing the mCherry or dsRedmito reporter for each well of the plate. Selected CD-14 positive monocytes incubated with GM-CSF for seven days showed changes in the cell morphology over the course of the differentiation period, going from spherical suspension cells to spindly and fully attached, and lastly, to more spread cells when fully differentiated.Fully differentiated cells were transected with the caspase BiFC pairs VC and VN along with the reporter plasmid dsRedmito, which was used to label the transected cells. Untreated cells showed no Venus fluorescence. And in nigericin treated cells, caspase-1 BiFC appeared as a single punctum with the typical shape of ASC specks.Quantitation of Venus-positive MDM showed that the highest percentage of caspase-1 BiFC is seen in the LPS plus nigericin treatment group. On plasmid titration of caspase-1, 4, and 5 pro-BiFC pairs, the highest percentage of Venus-positive cells resulted from 400, 500, and 1, 000 nanograms of transected plasmid, respectively. Confocal images obtained with a 20 times objective of a field of cells 24 hours post-transection showed that the untreated transected cells are viable as mitochondria are intact.After treatment with heme, the caspase-1 complex appears as a single green punctum similar to the one induced by nigericin. Remember to avoid harsh conditions and excessive manipulation of the cells during differentiation, transfection, and treatment as it can result in a spontaneous activation and high background levels of caspase activation.

visualization-inflammatory-caspases-induced-proximity-human-monocyte

Research

JoVE Journal

Immunology and Infection

2.5K Views.  Baylor College of Medicine. Questo protocollo è significativo perché può essere utilizzato per visualizzare e interrogare la via della caspasi infiammatoria a livello molecolare nelle cellule viventi in un particolare stato patologico. Il vantaggio principale di questa tecnica è che può essere utilizzata per identificare i componenti, i requisiti e la localizzazione dei complessi infiammatori di attivazione della caspasi nelle cellule immunitarie primarie. Con ottimizzazioni minori, questo metodo può essere adatta...