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 hav...

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.

<table><tbody><tr><td>0.25% pancreatic enzyme solution (excluding EDTA)</td><td>BOSTER Biological Technology co.ltd</td><td>PYG0068</td><td></td></tr><tr><td>5 mL Polystyrene Round-Bottom Tube</td><td>CORNING</td><td>352235</td><td></td></tr><tr><td>5 mL centrifuge tube</td><td>Labgic Technology Co., Ltd.&amp;nbsp;</td><td>BS-50-M</td><td></td></tr><tr><td>6-well plate&amp;nbsp;</td><td>Sorfa Life Science Research Co.,Ltd</td><td>220100</td><td></td></tr><tr><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&amp;times;Binding buffer, Apoptosis Positive Control Solution</td></tr><tr><td>celculture CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Esco (Shanghai) Enterprise Development Co., Ltd.</td><td>N/A</td><td></td></tr><tr><td>cell culture dish, 100 mm</td><td>Sorfa Life Science Research Co.,Ltd</td><td>230301</td><td></td></tr><tr><td>Cellometer K2 Fluorescent Cell Counter</td><td>Nexcelom Bioscience LLC</td><td>Cellometer K2</td><td></td></tr><tr><td>Cellometer SD100 Counting Chambers</td><td>Nexcelom Bioscience LLC</td><td>CHT4-SD100-002</td><td></td></tr><tr><td>centrifuge machine</td><td>Hunan Xiangyi Laboratory Instrument Development Co., Ltd</td><td>L530</td><td></td></tr><tr><td>chromium alum&amp;nbsp;</td><td>Guangdong Wengjiang Chemical Reagent Co., Ltd.</td><td>PA04354</td><td></td></tr><tr><td>cover glasses, 9 mm</td><td>Labgic Technology Co., Ltd.&amp;nbsp;</td><td>BS-09-RC</td><td></td></tr><tr><td>critical point dryer</td><td>Quorum Technologies</td><td>K850</td><td></td></tr><tr><td>dimethyl sulfoxide</td><td>BOSTER Biological Technology co.ltd</td><td>PYG0040</td><td></td></tr><tr><td>electron microscope fixative</td><td>Servicebio Technology co.ltd&amp;nbsp;</td><td>G1102</td><td>2.5% glutaric dialdehyde, 100 mM phosphorous salts</td></tr><tr><td>electronic balance</td><td>SHIMADZU</td><td>ATX124</td><td></td></tr><tr><td>ethanol absolute</td><td>Chengdu Kelong Chemical Co., Ltd</td><td>2021033102</td><td></td></tr><tr><td>flow cytometer</td><td>Becton,Dickinson and Company</td><td>FACSCanto &lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/66831/66831eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt;</td><td></td></tr><tr><td>flow cytometry analysis software</td><td>Becton,Dickinson and Company</td><td>BD FACSDiva&lt;sup&gt;TM&lt;/sup&gt; Software</td><td></td></tr><tr><td>gelatin</td><td>Guangdong Wengjiang Chemical Reagent Co., Ltd.</td><td>PA00256&amp;nbsp;</td><td></td></tr><tr><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&amp;amp;Technology Co.,Ltd.</td><td>CL0233</td><td></td></tr><tr><td>inverted microscope&amp;nbsp;</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&amp;nbsp;</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd.&amp;nbsp;</td><td>L8880</td><td>LPS is derived from Escherichia coli 055:B5</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;ATP</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd.&amp;nbsp;</td><td>A8270</td><td></td></tr><tr><td>phorbol-12-myristate-13-acetate&amp;nbsp;</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd.&amp;nbsp;</td><td>P6741</td><td></td></tr><tr><td>phosphate-buffered saline</td><td>Servicebio Technology co.ltd&amp;nbsp;</td><td>G4202</td><td></td></tr><tr><td>Pipette</td><td>Eppendorf AG</td><td>N/A</td><td></td></tr><tr><td>pipette tips, 10 &amp;mu;L</td><td>Servicebio Technology co.ltd&amp;nbsp;</td><td>T-10PL</td><td></td></tr><tr><td>pipette tips, 1 mL&amp;nbsp;</td><td>Servicebio Technology co.ltd&amp;nbsp;</td><td>T-1250L</td><td></td></tr><tr><td>pipette tips, 200 &amp;mu;L</td><td>Servicebio Technology co.ltd&amp;nbsp;</td><td>T-200L</td><td></td></tr><tr><td>RPMI-1640 complete culture media</td><td>Procell Life Science&amp;amp;Technology Co.,Ltd.</td><td>CM0233</td><td>RPMI-1640 + 10% FBS + 0.05mM &amp;beta;-mercaptoethanol + 1% P/S</td></tr><tr><td>RPMI-1640 culture media&amp;nbsp;</td><td>Shanghai&amp;nbsp;BasalMedia&amp;nbsp;Technologies Co., LTD.</td><td>K211104</td><td></td></tr><tr><td>sheath fluid</td><td>BECKMAN COULTER</td><td>8546733</td><td></td></tr><tr><td>sputter coater</td><td>Cressington Scientific Instruments Ltd</td><td>108</td><td></td></tr><tr><td>thermostatic water bath</td><td>GUOHUA Electric Appliance CO.,Ltd&amp;nbsp;</td><td>HH-1</td><td></td></tr></tbody></table>

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 ...

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

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

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Immunology and Infection

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles (MPs) are one such type of vesicles, ranging in diameter from 0.1 &#956;m to 1 &#956;m, and typically shed from the plasma membrane of eukaryotic cells undergoing activation or apoptosis. Here we describe the generation of T lymphocyte&#8211;derived microparticles (LMPs) from apoptotic CEM T cells stimulated with actinomycin D. LMPs are isolated through a multistep differential centrifugation process and characterized using flow cytometry. This protocol also presents an in situ cell death detection method for demonstrating the proapoptotic effect of LMPs on bronchial epithelial cells derived from mouse primary respiratory bronchial tissue explants. Methods described herein provide a reproducible procedure for isolating abundant quantities of LMPs from apoptotic lymphocytes in vitro. LMPs derived in this manner can be used to evaluate the characteristics of various disease models, and for pharmacology and toxicology testing. Given that the airway epithelium offers a protective physical and functional barrier between the external environment and underlying tissue, use of bronchial tissue explants rather than immortalized epithelial cell lines provides an effective model for investigations requiring airway tract tissue.

Cell membrane&#8211;shed microparticles (MPs) are active biological vesicles that can be isolated and their pathophysiological effects investigated in various models. Here we describe a method for generating MPs derived from T lymphocytes (LMPs) and for demonstrating their proapoptotic effect on airway epithelial cells.

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development and implementation of biomedical devices and tissue engineering constructs. Macrophages, an innate immune cell, are key players in the foreign body reaction because they remain at the implant site for the lifetime of the device, and are commonly studied to gain an understanding of this detrimental host response. Many biomaterials researchers have shown that adsorbed protein layers on implanted materials influence macrophage behavior, and subsequently impact the host response. The methods in this paper describe an in vitro model using adsorbed protein layers containing cellular damage molecules on polymer biomaterial surfaces to assess macrophage responses. An NF-&#1082;B/AP-1 reporter macrophage cell line and the associated colorimetric alkaline phosphatase assay were used as a rapid method to indirectly examine NF-&#1082;B/AP-1 transcription factor activity in response to complex adsorbed protein layers containing blood proteins and damage-associated molecular patterns, as a model of the complex adsorbed protein layers formed on biomaterial surfaces in vivo.

This protocol provides researchers with a rapid, indirect method of measuring TLR-dependent NF-&#1082;B/AP-1 transcription factor activity in a murine macrophage cell line in response to a variety of polymeric surfaces and adsorbed protein layers that model the biomaterial implant microenvironment.

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development ...

Bioengineering

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

Medicine

Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line

Tumor-associated macrophages (TAM) can switch their expression and cytokine profile according to external stimuli. This remarkable plasticity enables TAM to adapt to ongoing changes within the tumor microenvironment. Macrophages can have either primarily pro-inflammatory (M1-like) or anti-inflammatory (M2-like) attributes and can continually switch between these two main states. M2-like macrophages within the tumor environment are associated with cancer progression and poor prognosis in several types of cancer. Many different methods for inducing differentiation and polarization of THP-1 cells are used to investigate cellular and intercellular mechanisms and the effects of TAM within the microenvironment of tumors. Currently, there is no established model for M2-like macrophage polarization using the THP-1 cell line, and the results of expression and cytokine profiles of macrophages due to certain in vitro stimuli vary between studies. This protocol serves as detailed guidance to differentiate THP-1 monocyte-like cells into M0 macrophages and to further polarize cells into an M2-like phenotype within 14 days. We demonstrate the morphological changes of THP-1 monocyte-like cells, differentiated macrophages, and polarized M2-like macrophages using light microscopy. This model is the basis for cell line models investigating the anti-inflammatory effects of TAM and their interactions with other cell populations of the tumor microenvironment.

M2-like tumor-associated macrophages (TAM) are associated with tumor progression and poor prognosis in cancer. This protocol serves as a detailed guide to reproducibly differentiate and polarize THP-1 monocyte-like cells into M2-like macrophages within 14 days. This model is the basis to investigate the anti-inflammatory effects of TAM within the tumor microenvironment.

Tumor-associated macrophages (TAM) can switch their expression and cytokine profile according to external stimuli. This remarkable plasticity enables TAM ...

Evaluation of Caspase Activation to Assess Innate Immune Cell Death

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

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

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

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

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Bone Marrow-derived Macrophage Production

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line

Evaluation of Caspase Activation to Assess Innate Immune Cell Death