We express our great appreciation to Jiayi Sun and Lu Yang at Innovative Institute of Chinese Medicine and Pharmacy, Chengdu University of Traditional Chinese Medicine, for the assistance with flow cytometry, Cuiping Chen at State Key Laboratory of Southwestern Chinese Medicine Resources, for the help with scanning electron microscopy. This work was supported by the National Natural Science Foundation of China [82104491], the Natural Science Foundation of Sichuan [2023NSFSC0674], and the Post-doctoral Science Foundation of China [2021M693789].

1. Cell line and cell culture
<ol>
	<li>Grow the human monocytic cell line THP-1 in RPMI-1640 complete culture medium with 10% fetal bovine serum, 1% penicillin-streptomycin and 0.05 mM &#946;-mercaptoethanol. Culture the cells at 37 &#176;C in 5% CO2 humidified air. Sub-culture cells every 2-3 days. 
	NOTE: THP-1 is a suspension cell, select to change half of the medium for sub-culturing. When observing many cell fragments under the light microscope, centrifuge at 300 x g for 3 min at room temperature. Resuspend cells in the pre-warmed fresh complete cell culture medium.</li>
	<li>Use cells (in passages 4-10) at the logarithmic growth phase for all experiments. When the cells are in good condition and logarithmic growth phase, they are round, bright, dense and without clumps and cell fragments under the light microscope at 100 x.</li>
</ol>
2. Differentiation of THP-1 cells
<ol>
	<li>Prepare a stock solution of PMA with a concentration of 1 mM (dissolve 1 mg of PMA in 1.62 mL of dimethyl sulfoxide). Dilute the PMA stock solution withcomplete cell culture medium to a final working concentration of 100 nM.</li>
	<li>Collect the cells in the culture dish into a 10 mL sterile centrifuge tube. Centrifuge at 300 x g for 3 min at room temperature, resuspend cells with 3 mL of complete culture medium containing PMA.</li>
	<li>Open the cell counter and the counting software. Pipette 20 &#181;L of cell suspension to the cell counter board and insert it into the cell counter board. Click Preview B1, turn the knob at the bottom right of the instrument, and adjust the focal distance to make the cells with bright center with clear contours on the software. Click Count to start counting.</li>
	<li>Based on the counting results, use complete culture medium containing PMA to adjust a cell density to 1 x 106 cells/mL. Seed cells in a 6 well plate, incubate the plate for 24 h at 37 &#176;C and 5% CO2 before starting further treatment.</li>
</ol>
3. Treatment of THP-1cells
<ol>
	<li>Prepare a stock solution of LPS with a concentration of 1 mg/mL (dissolve 1 mg of LPS in 1 mL of phosphate-buffered saline (PBS)). Dilute the LPS stock solution with serum-free medium to a final working concentration of 1 &#181;g/mL. Prepare a stock solution of Na2ATP with a concentration of 500 mM (dissolve 0.3026 g of ATP in 1 mL of PBS).</li>
	<li>Aspirate the PMA-containing medium from wells and wash with PBS 1x. Add serum-free medium to the control group and serum-free medium containing LPS to the model group. Incubate the plate for 6 h at 37 &#176;C + 5% CO2 before proceeding with further treatment.</li>
	<li>After 6 h, add the ATP stock solution to the model group to a final working concentration of 500 nM. Incubate the plate for 45 min at 37 &#176;C + 5% CO2 before proceeding.</li>
</ol>
4. Flow cytometry analysis (Method 1)
<ol>
	<li>Preparation of experimental samples
	<ol>
		<li>Seed and incubate cells in 6-well plates according to step 2. Establish four control groups with no treatment: one no-staining control, two single-staining controls (one for each stain), and one double-staining control. Establish one treatment group and treat according to step 3.</li>
		<li>After treatment, aspirate all the supernatant from the wells and wash with PBS 2x. Add 500 &#181;L of 0.05% Trypsin (non-EDTA) to each well, and incubate in the incubator for 30 s.</li>
		<li>Add 1 mL of RPMI-1640 complete culture medium to each well to stop cell detaching and collect the cells into the 5 mL tube. Centrifuge at 300 x g for 3 min at room temperature.</li>
		<li>Add 1 mL of PBS to resuspend cells. Place tubes with cells in a water bath at 37 &#176;C for 3 min. Centrifuge all samples at 300 x g for 3 min at room temperature.</li>
		<li>Dilute 200 &#181;L of 5x binding buffer with 800 &#181;L of distilled water to 1x binding buffer. Add 100 &#181;L of 1x binding buffer to resuspend cells and transfer the cells into the 5 mL tube.</li>
		<li>Add 5 &#181;L of Annexin V-PE solution to one tube of control group cells for 10 min and 5 &#181;L of 7-AAD solution to another tube of control group cells for 5 min (as single staining controls separately). Gently vortex each tube to mix dye and incubate the samples at room temperature protected from light. Add 400 &#181;L of PBS to each tube and vortex gently to terminate incubation.</li>
		<li>Add 5 &#181;L of Annexin V-PE solution to the remaining tubes and incubate for 5 min at room temperature. Gently vortex each tube to mix dye and incubate the samples at room temperature protected from light. After 5 min, add 5 &#181;L of 7-AAD solution for 5 min at room temperature. Add 400 &#181;L of PBS to each tube and vortex gently to terminate the incubation.</li>
		<li>Filter the cell suspension using a 35 &#181;m nylon mesh that are part of the 5 mL polystyrene round-bottom tubes. Place the tube on ice and wait for testing. Gently mix before testing and then insert it into the instrument.</li>
	</ol>
	</li>
	<li>Flow cytometry
	<ol>
		<li>Use fluorescence-activated cell sorting flow cytometer for testing. Open the flow cytometer data analysis software and confirm the performance of the detector and laser. Click the Cytometer and choose Fluidics startup, wait for the system to be ready. Exclude flow chamber bubbles by clicking on Cytometer &#62; Clean Modes &#62; De-gas Flow cell.</li>
		<li>Click Experiment, select in sequence New folder, Experiment, Specimen, Tube and Edit Name. A pentagonal shaped arrow in front of the tube, turns green when light up.</li>
		<li>Click on the icon of Cytometer FACSCelesta (a shortcut key icon), select Parameter, and then select FSC, SSC and PE signal. Click on the icon of Global Worksheet (a shortcut key icon) to establish PE histogram. For the flow cytometer used in this study, use PE for Annexin V at 586 nm and plot on the X-axis; PerCP-Cy5.5 for 7-AAD at 700 nm and plot on the Y-axis.</li>
		<li>Insert unstained control sample first into the instrument. Click on the icon of Acquisition Dashboard (a shortcut key icon), acquire a minimum of 10,000 events from the desired population. Exclude debris using a gate (P1) on an FSC-A and SSC-A dot plot. Adjust flow rate to Middle speed and Record Data. Adjust voltage to place cell population on the diagonal of the FSC/SSC histogram and click Restart. Adjust flow rate to Middle speed and Record Data.</li>
		<li>Click Next tube, run single staining controls of Annexin V and 7-AAD to adjust compensation. Run the remaining tubes and collect data.</li>
	</ol>
	</li>
</ol>
5. SEM imaging (Method 2)
<ol>
	<li>Preparation of experimental samples
	<ol>
		<li>Seed and incubate cells in 6-well plates according to step 2 and treat according to step 3.</li>
		<li>After treatment, aspirate all the supernatant from the wells and wash 2x with PBS. Add 500 &#181;L of 0.05% Trypsin (non-EDTA) to each well, and incubate in the incubator for 30 s.</li>
		<li>Add 1 mL of RPMI-1640 complete culture medium to each well to stop cell detaching and collect the cells into the 5 mL tube. Centrifuge at 300 x g for 3 min at room temperature.</li>
		<li>Fix cells with 500 &#181;L of electron microscope fixative (2.5% glutaric dialdehyde, 100 mM phosphorous salts) for 2 h at room temperature, followed by overnight at 4 &#176;C.</li>
		<li>Prepare chromium alum solution: Add 1.0 g of gelatin to 100 mL of ultrapure water, 70 &#176;C water-bath heating, and stir evenly. Add 0.05 g of chromium alum, stir evenly and filter with filter paper. Dip the clean cover glass in chromium alum solution for 2 h at 37 &#176;C and dry it in a 37 &#176;C oven for later use. 
		NOTE: The purpose is to prevent cell detachment during the experiment.</li>
		<li>Collect cells from fixed solution. Centrifuge at 300 x g for 3 min at room temperature. Add 100 &#181;L of PBS and gently blow away the cells. Drop the cell suspension onto the cover glass, allow to stand for 5 min. Aspirate all the supernatant and wash 3x with PBS for 5 min each time. 
		NOTE: The operation must be performed gently to prevent cell detachment. Do not shake vigorously, move slowly, and add liquid slowly along the wall.</li>
		<li>Add 500 &#956;L of 1% osmic&#160;acid fixation for 1 hour. Aspirate all the&#160;supernatant and wash 3x with PBS for 5 min&#160;each time.</li>
		<li>Dehydrate the samples successively in a series of ethanol (EtOH) concentrations (30%, 50%, 70%, 80%, 90%, 95%, 100%, 100%) for 15 min per EtOH solution. 
		NOTE: The cover glasses can&#160;be kept in 100% EtOH for several days at 4 &#176;C&#160;but it should dry as soon as possible to avoid&#160;excessive dehydration.</li>
		<li>Switch on the critical point dryer, open the CO2 tank, cool the chamber to 10 &#176;C. Quickly wrap the sample with filter paper when the chamber pressure is 0 psi and put it into the chamber.</li>
		<li>Open the inlet valve, observe through CO2 observation window, raise the liquid CO2 level between the two red lines (Do not exceed the upper limit), close the inlet valve. Soak the sample for 10 min. Open the inlet and exhaust valves simultaneously to equalize the CO2 for 1 min (liquid CO2 displaces EtOH). Close the exhaust valve to raise the liquid CO2 level to the specified position, close the inlet valve.</li>
		<li>Set the temperature to 35 &#176;C and pressure at 1250 psi. Once the temperature and pressure stabilize, depressurize at 100 psi/min. Take out the sample when the chamber pressure is zero and switch off the instrument.</li>
		<li>Mount specimens on SEM aluminum specimen holders using conductive tape. Employ a sputter coater tocover the samples with a layer (2-5 nm) of gold.</li>
	</ol>
	</li>
	<li>Imaging
	<ol>
		<li>Use high resolution cold field emission SEM for imaging. Set the accelerating voltage of SEM to 3 kV. Set the working distance to 10 mm. Set the magnification to 1,000x to observe the panorama. Set the magnification to 5,000x and 20,000x to locate the plasma membrane and image the sample. 
		NOTE: The process of visualizing PMA-differentiated THP-1 macrophages via SEM is similar to other types of cells and tissues and depends on the instruments used. The reference14 provides a detailed SEM protocol with accompanying videos.</li>
	</ol>
	</li>
</ol>

Analyzing LPS/ATP-Triggered Cell Death Mechanisms in THP-1 Cells

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The authors have nothing to disclose.

In this manuscript, two methods were used to detect LPS and ATP-induced death of PMA-differentiated THP-1 macrophages. Annexin V/7-AAD double staining was used, and the results were analyzed by flow cytometry from overall staining. As with other flow cytometric analysis, a group of unstained cells and two groups of single-stained cells were set up to exclude false positive and false negative results. The results show that after LPS/ATP stimulation, several cells lost membrane integrity, indicating that cell death may have occurred. This detection method allows us to evaluate the cell death from whole cells. It is worth noting that the Annexin V/7-AAD double staining is not sufficient to distinguish between pyroptosis, apoptosis, and necroptosis. In the early stages of these three forms, PS will be exposed outside the plasma membrane, and Annexin V staining is positive. The integrity of membrane is lost during the late stage of these three forms, and the staining results show double-positive staining. Therefore, we used SEM to further observe the possible forms of cell death that might occur.
In apoptosis, BAX/BAK pore formation on the mitochondrial outer membrane, subsequent cell shrinkage and cell membrane blebbing occurs. Necroptosis employs MLKL to open pores in the cell membrane, while pyroptosis signal activates gasdermin to form pores in the cell membrane. It is worth noting that both pyroptosis and necroptosis are accompanied by cell membrane rupture in the final stage. However, gasedermin pores are much larger than MLKL pores, so gasdermin pores do not have ion selectivity. Necroptotic cells exhibit significant swelling in morphology15. They ultimately end in membrane rupture. Regardless of the signaling pathway upstream of cell membrane permeability activation, changes in the cell membrane accompany these three forms of cell death. We used SEM to further observe the cell membrane, which provide an intuitive way to examine cell death. We observed the characteristics of pyroptosis, including membrane blebbing and the rupture membrane, and necroptosis, including cell swelling and the rupture membrane, consistent with characteristics studied by previous researchers15. We observed membrane blebbing and cell shrinkage, consistent with the characteristics of apoptosis studied by previous researchers16.
In previous studies, the combination of LPS and ATP was often used to induce pyroptosis. Interestingly, we observed features of apoptosis and necroptosis. Although all three forms may occur in LPS/ATP-induced cell death, previous studies have shown that pyroptosis remains the dominant mode of death. LPS is one of the most characterized pathogen-associated molecular patterns. ATP released from dying cells, has a high concentration within the cell. Extracellular ATP is one of the danger-associated molecular patterns17. LPS/ATP is a classic two-step stimulation method to induce pyroptosis. Research has shown that the addition of ATP to LPS-stimulated cells can rapidly activate caspase-118 while silencing the caspase-1 gene can significantly reverse LPS/ATP-induced cell death19. In previous studies, we have also found LPS/ATP-induced activation of apoptotic and necroptotic target proteins20,21. Although LPS/ATP is a classic pyroptosis inducer, it may also induce apoptosis and necroptosis.
In fact, these three forms often do not exist independently, and crosstalk between signal stages. For example, gasdermin proteins target the mitochondria to promote cytochrome c release to enhance the mitochondrial apoptotic pathway22. Activation of caspase-3 can cleave gasdermin E and then trigger pyroptosis23. A study suggests that MLKL/PIPK3 signaling is involved in the activation of NLRP3 inflammasomes24. This complex cross-talk led to the concept of PANoptosis, defined as an inflammatory programmed cell death pathway regulated by the PANoptosome complex with key features of pyroptosis, apoptosis, and/or necroptosis that cannot be accounted for by any of these programmed cell death pathways alone25. As the concept of PANoptosis was proposed, scientists described the occurrence of programmed cell death more comprehensively, this also highlights the complexity of programmed cell death. Meanwhile, this also indicates the limitations of this research method. Although pyroptosis is considered dominant in LPS/ATP-induced cell death, one cannot determine the proportion of these death forms or whether they all exist, even involving other forms of programmed death. This can be an area to investigate in the future.
This protocol is based on LPS and ATP-induced death of macrophages. We have described a method of testing from the whole to the individual, which is also applicable to other models and cells. The results indicate that cells may have undergone pyroptosis, apoptosis, or necroptosis, which is not limited to pyroptosis, helping researchers better understand cell death after LPS and ATP stimulation and choose a better experimental method. In previous studies, Annexin V/7-AAD or Annexin V/PI double staining was commonly used to indicate the occurrence of apoptosis26, 27, some researchers have also used it to study apoptosis28. In fact, we cannot distinguish between the three types of cell death through these double staining. This protocol reminds researchers, especially beginners, that Annexin V/7-AAD or Annexin V/PI double staining cannot qualitative the type of cell death. It can only show cell damage and death as a whole or indicate that the cell may have undergone some form of death. In more targeted research, it is necessary to introduce a control group that inhibits other deaths to show that conclusively program death is at hand. Similarly, further mechanistic research will investigate the critical targets of different programmed deaths.
In addition, during the preparation of the experiment, we found that there are currently many detailed experimental procedures for SEM of adherent cells, with little description of suspended cells. Here, we provide a more detailed description of the sample preparation method for suspended cells. Step 5.5 is a necessary step to assist in immobilizing suspended cells onto cell slides. Subsequent operations must be performed gently to prevent cell detachment. In addition to the chromium alum solution, poly-L-lysine can also immobilize cells.

Cell death is a fundamental physiological process in all living organisms. In recent years, substantial studies have shown that cell death is involved in the immunity and balance within the organism. Studying cell death helps us better understand the onset and development of diseases. Several forms of programmed cell death have been described, and some key targets in these processes have been identified. Pyroptosis, apoptosis, and necroptosis are the three genetically defined programmed cell death pathways involved in internal balance and disease1.
Pyroptosis is characterized by the formation of membrane pores and the release of cell contents. Activated caspases or granzymes cleave gasdermins to separate their N-terminal domains, which then oligomerize, bind to the membrane, and form pores2,3,4. The gasdermin pore provides an atypical secretion channel across cellular membranes, resulting in downstream cell responses, including content release and ion influx2,3,4. Ultimately, the cells eventually experience plasma membrane rupture and pyroptotic lysis facilitated by ninjurin-15. In apoptosis, activated Bax and Bak form oligomers on the mitochondrial outer membrane and release cytochrome C, which is regulated by a balance between proapoptotic and antiapoptotic proteins of the BCL-2 family, initiator caspases (caspase-8, -9 and -10) and effector caspases (caspase-3, -6 and -7)1,6,7. The morphological changes of apoptosis include membrane blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and apoptotic body formation6,8. The receptor-interacting serine/threonine-protein kinase 1 (RIPK3) and mixed-lineage kinase domain-like (MLKL) are two downstream core components of the necroptotic mechanism1. RIPK3 recruits and phosphorylates MLKL, p-MLKL oligomerizes, associates with the cell membrane, initiates membrane perforations, causes ion influx, increases intracellular osmolarity, and eventually cell rupture6,9. Gasdermins and MLKL bind to the plasma membrane and mediate pyroptosis and necroptosis, respectively, while BAX/BAK mediates apoptosis by binding to the outer membrane of mitochondria6.
Although each pathway has specific mechanisms and outcomes, they lead to similar changes on the cell membrane. Under normal circumstances, phosphatidylserine (PS) is only localized in the inner leaflet of the plasma membrane. However, in the early stages of pyroptosis, apoptosis, and necroptosis, PS will be exposed, outside of the plasma membrane. Caspase-3/caspase-7 activates TMEM16 and XKR families, which leads to an asymmetrical cell membrane and externalizes&#160;PS during apoptosis10. Gasdermin D-mediated and MLKL-mediated Ca2+ influx leads to loss of the symmetry of the phospholipid bilayer on the cell membrane and the exposure of PS. The exposure occurs before the loss of cell membrane integrity11,12. Based on the similar changes in the membrane of these three types of programmed cell death, we use flow cytometry to analyze Annexin V/7-Aminoactinomycin D (7-AAD) double staining to detect cell death. Annexin V, a calcium-dependent phospholipid-binding protein, has a high affinity for PS, which can serve as a sensitive probe to detect exposed PS on the surface of the plasma membrane13. 7-AAD is a nucleic acid stain that cannot pass through the entire cell membrane. It is similar to propidium iodide (PI), a commonly used nucleic acid dye. They have similar fluorescence characteristics, but 7-AAD has a narrower emission spectrum and less interference with other detection channels. Owing to these similarities, it is not sufficient to distinguish between pyroptosis, apoptosis, and necroptosis. We used flow cytometry to detect the cell death from whole cells. A second method is used to capture the cell membrane using scanning electron microscopy (SEM) to observe the possible forms of cell death in individual cells.
We established a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced phorbol-12-myristate-13-acetate (PMA)-differentiated lipid deposition in human monocyte (THP-1) macrophage model to observe cell death. This protocol focuses on observing cell death rather than investigating mechanisms.

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			<td>0.25% pancreatic enzyme solution (excluding EDTA)</td>
			<td>BOSTER Biological Technology co.ltd</td>
			<td>PYG0068</td>
			<td></td>
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			<td>5 mL Polystyrene Round-Bottom Tube</td>
			<td>CORNING</td>
			<td>352235</td>
			<td></td>
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			<td>5 mL centrifuge tube</td>
			<td>Labgic Technology Co., Ltd.&#160;</td>
			<td>BS-50-M</td>
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			<td>6-well plate&#160;</td>
			<td>Sorfa Life Science Research Co.,Ltd</td>
			<td>220100</td>
			<td></td>
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			<td>Annexin V-PE/7-AAD apoptosis analysis kit</td>
			<td>Absin (Shanghai) Biological Technology co.ltd</td>
			<td>abs50007</td>
			<td>Annexin V-PE, 7-AAD, 5&#215;Binding buffer, Apoptosis Positive Control Solution</td>
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			<td>celculture CO2 incubator</td>
			<td>Esco (Shanghai) Enterprise Development Co., Ltd.</td>
			<td>N/A</td>
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			<td>cell culture dish, 100 mm</td>
			<td>Sorfa Life Science Research Co.,Ltd</td>
			<td>230301</td>
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			<td>Cellometer K2 Fluorescent Cell Counter</td>
			<td>Nexcelom Bioscience LLC</td>
			<td>Cellometer K2</td>
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			<td>Cellometer SD100 Counting Chambers</td>
			<td>Nexcelom Bioscience LLC</td>
			<td>CHT4-SD100-002</td>
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			<td>centrifuge machine</td>
			<td>Hunan Xiangyi Laboratory Instrument Development Co., Ltd</td>
			<td>L530</td>
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			<td>chromium alum&#160;</td>
			<td>Guangdong Wengjiang Chemical Reagent Co., Ltd.</td>
			<td>PA04354</td>
			<td></td>
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			<td>cover glasses, 9 mm</td>
			<td>Labgic Technology Co., Ltd.&#160;</td>
			<td>BS-09-RC</td>
			<td></td>
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			<td>critical point dryer</td>
			<td>Quorum Technologies</td>
			<td>K850</td>
			<td></td>
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			<td>dimethyl sulfoxide</td>
			<td>BOSTER Biological Technology co.ltd</td>
			<td>PYG0040</td>
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			<td>electron microscope fixative</td>
			<td>Servicebio Technology co.ltd&#160;</td>
			<td>G1102</td>
			<td>2.5% glutaric dialdehyde, 100 mM phosphorous salts</td>
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			<td>electronic balance</td>
			<td>SHIMADZU</td>
			<td>ATX124</td>
			<td></td>
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			<td>ethanol absolute</td>
			<td>Chengdu Kelong Chemical Co., Ltd</td>
			<td>2021033102</td>
			<td></td>
		</tr>
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			<td>flow cytometer</td>
			<td>Becton,Dickinson and Company</td>
			<td>FACSCanto <img alt="Equation 1" src="/files/ftp_upload/66831/66831eq01.jpg" style="margin: auto;" /></td>
			<td></td>
		</tr>
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			<td>flow cytometry analysis software</td>
			<td>Becton,Dickinson and Company</td>
			<td>BD FACSDivaTM Software</td>
			<td></td>
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			<td>gelatin</td>
			<td>Guangdong Wengjiang Chemical Reagent Co., Ltd.</td>
			<td>PA00256&#160;</td>
			<td></td>
		</tr>
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			<td>High resolution cold field emission scanning electron microscope</td>
			<td>TITACHI</td>
			<td>Regulus 8100</td>
			<td></td>
		</tr>
		<tr>
			<td>human monocytic cell line THP-1</td>
			<td>Procell Life Science&#38;Technology Co.,Ltd.</td>
			<td>CL0233</td>
			<td></td>
		</tr>
		<tr>
			<td>inverted microscope&#160;</td>
			<td>Leica Microsystems Co., Ltd</td>
			<td>DMi1</td>
			<td></td>
		</tr>
		<tr>
			<td>IR Vortex Mixer</td>
			<td>VELP Scientifica Srl</td>
			<td>ZX4</td>
			<td></td>
		</tr>
		<tr>
			<td>lipopolysaccharide&#160;</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>L8880</td>
			<td>LPS is derived from Escherichia coli 055:B5</td>
		</tr>
		<tr>
			<td>Na2ATP</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>A8270</td>
			<td></td>
		</tr>
		<tr>
			<td>phorbol-12-myristate-13-acetate&#160;</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>P6741</td>
			<td></td>
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			<td>phosphate-buffered saline</td>
			<td>Servicebio Technology co.ltd&#160;</td>
			<td>G4202</td>
			<td></td>
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			<td>Pipette</td>
			<td>Eppendorf AG</td>
			<td>N/A</td>
			<td></td>
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			<td>pipette tips, 10 &#956;L</td>
			<td>Servicebio Technology co.ltd&#160;</td>
			<td>T-10PL</td>
			<td></td>
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			<td>pipette tips, 1 mL&#160;</td>
			<td>Servicebio Technology co.ltd&#160;</td>
			<td>T-1250L</td>
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			<td>pipette tips, 200 &#956;L</td>
			<td>Servicebio Technology co.ltd&#160;</td>
			<td>T-200L</td>
			<td></td>
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			<td>RPMI-1640 complete culture media</td>
			<td>Procell Life Science&#38;Technology Co.,Ltd.</td>
			<td>CM0233</td>
			<td>RPMI-1640 + 10% FBS + 0.05mM &#946;-mercaptoethanol + 1% P/S</td>
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		<tr>
			<td>RPMI-1640 culture media&#160;</td>
			<td>Shanghai&#160;BasalMedia&#160;Technologies Co., LTD.</td>
			<td>K211104</td>
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			<td>sheath fluid</td>
			<td>BECKMAN COULTER</td>
			<td>8546733</td>
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			<td>sputter coater</td>
			<td>Cressington Scientific Instruments Ltd</td>
			<td>108</td>
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			<td>thermostatic water bath</td>
			<td>GUOHUA Electric Appliance CO.,Ltd&#160;</td>
			<td>HH-1</td>
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lps and atp-induced death of pma-differentiated thp-1 macrophages and its validation

Cell death is a fundamental process in all living organisms. The protocol establishes a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced phorbol-12-myristate-13-acetate (PMA)-differentiated lipid deposition in human monocyte (THP-1) macrophage model to observe cell death. LPS combined with ATP is a classic inflammatory induction method, often used to study pyroptosis, but apoptosis and necroptosis also respond to stimulation by LPS/ATP. Under normal circumstances, phosphatidylserine is only localized in the inner leaflet of the plasma membrane. However, in the early stages of pyroptosis, apoptosis, and necroptosis, the cell membrane remains intact and exposed to phosphatidylserine, and in the later stages, the cell membrane loses its integrity. Here, flow cytometry was used to analyze Annexin V and 7-Aminoactinomycin D (AAD) double staining to detect the cell death from the whole cells. The results show&#160;that substantial cells died after stimulation with LPS/ATP. Using scanning electron microscopy, we observe the possible forms of cell death in individual cells. The results indicate that cells may undergo pyroptosis, apoptosis, or necroptosis after stimulation with LPS/ATP. This protocol focuses on observing the death of macrophages after stimulation with LPS/ATP. The results showed that cell death after LPS and ATP stimulation is not limited to pyroptosis and that apoptosis and necrotic apoptosis can also occur, helping researchers better understand cell death after LPS and ATP stimulation and choose a better experimental method.

The cell samples were treated as described in the protocol and flow cytometry detection was done. Normal cells cannot be stained with Annexin V and 7-AAD (Annexin V-/7-AAD-). In the early stages of pyroptosis, apoptosis, and necroptosis, PS was exposed and bound to Annexin V, but the cell membrane was still intact and excluded 7-AAD from the extracellular space (Annexin V+/7-AAD-). In the later stages, the cell membrane loses its integrity, cells are simultaneously stained by Annexin V and 7-AAD, showing double positive results (Annexin V+/7-AAD+). The necrotic cells were only stained with 7-AAD (Annexin V-/7-AAD+). Figure 1 shows that after LPS/ATP stimulation, the cells in the Q2 region increased significantly, indicating cell death.
We use SEM to capture the plasma membrane surface, directly observing the characteristics of membrane from a single cell. Figure 2A shows the control cells, Figure 2B-D show the cells after LPS/ATP stimulation. We observed cells with characteristics of pyroptosis, including membrane blebbing and rupture membrane (Figure 2B), cells with apoptotic characteristics, including membrane blebbing and cell shrinkage (Figure 2C), and cells with necroptosis characteristics, including cell swelling and rupture membrane (Figure 2D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66831/66831fig01.jpg" /> 
Figure 1: Detection of Annexin V/7-AAD double staining using flow cytometry. (A) Annexin V negative and 7-AAD negative staining (Q4), Annexin V positive and 7-AAD negative double staining (Q3), Annexin V positive and 7-AAD positive staining (Q2), and Annexin V negative and 7-AAD positive staining (Q1). (B) Quantitative data for (A). Data are presented as mean &#177; SD (n=3), unpaired t test, **p &#60; 0.01. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66831/66831fig01large.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/66831/66831fig02.jpg" /> 
Figure 2: Scanning electron microscopy images of PMA-differentiated THP-1 macrophages. (A) Control group, 5,000x/20,000x magnification. (B, C, D) LPS/ATP group show membrane blebbing, rupture of membrane (&#8593;), cell swelling (&#8593;), and cell shrinkage (&#8593;) at 5,000x/20,000x magnification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66831/66831fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Read this Scientific Article Protocol about Author Spotlight: THP-1 Macrophage Response to LPS/ATP - Unveiling Pyroptosis, Apoptosis and Necroptosis Spectrum at JoVE.com

Author Spotlight: THP-1 Macrophage Response to LPS/ATP &#8212; Unveiling the Pyroptosis, Apoptosis, and Necroptosis Spectrum

This protocol is based on LPS and ATP-induced death of PMA-differentiated THP-1 macrophages. We use flow cytometry to analyze Annexin V and 7-AAD double staining to detect cell death, using the whole cell and employing scanning electron microscopy to observe the cell membrane morphology.

LPS and ATP-induced Death of PMA-differentiated THP-1 Macrophages and its Validation

Cell death is a fundamental process in all living organisms. The protocol establishes a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced ...

lps-atp-induced-death-pma-differentiated-thp-1-macrophages-its

Nanyang Technological University

Research

JoVE Journal

Biology

1.5K Views.  Chengdu University of Traditional Chinese Medicine. This protocol is based on LPS and ATP-induced death of PMA-differentiated THP-1 macrophages. We use flow cytometry to analyze Annexin V and 7-AAD double staining to detect cell death, using the whole cell and employing scanning electron microscopy to observe the cell membrane morphology.

Video: Author Spotlight: THP-1 Macrophage Response to LPS/ATP — Unveiling the Pyroptosis, Apoptosis, and Necroptosis Spectrum

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

A &#946;-glucuronidase (GUS) Based Cell Death Assay

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative positive or negative regulators of cell death. In this system, N. benthamiana leaves are co-infiltrated with a 35S driven expression cassette containing the gene to be analyzed, and the GUS vector pCAMBIA 2301 using Agrobacterium strain LBA4404 as a vehicle. Because live cells are required for GUS expression to occur, loss of GUS activity is expected when this marker gene is co-expressed with positive regulators of cell death. Equally, increased GUS activity is observed when anti-apoptotic genes are used compared to the vector control. As shown below, we have successfully used this system in our lab to analyze both pro- and anti-death players. These include the plant anti-apoptotic Bcl-2 Associated athanoGene (BAG) family, as well as, known mammalian inducers of cell death, such as BAX. Additionally, we have used this system to analyze the death function of specific truncations within proteins, which could provide clues on the possible post-translational modification/activation of these proteins. Here, we present a rapid and sensitive plant based method, as an initial step in investigating the death function of specific genes.

A &#946;-glucuronidase GUS Based Cell Death Assay

Programmed cell death assays commonly used in mammalian systems such as DNA laddering or TUNEL assays, are often difficult to reproduce in plants. In combination with a GUS reporter system, we propose a rapid, plant based transient assay to analyze the potential death properties of specific genes.

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 &#946;-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called &#34;domainome&#34;, will be well expressed and soluble, making them suitable for structural/functional studies.
By cloning and displaying the &#34;domainome&#34; directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification.
The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.

The protocols described allow the construction, characterization and selection (against the target of choice) of a &#34;domainome&#34; library made from any DNA source. This is achieved by a research pipeline that combines different technologies: phage display, a folding reporter and next generation sequencing with a web tool for data analysis.

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

LPS and ATP-induced Death of PMA-differentiated THP-1 Macrophages and its Validation

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Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages