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

Summary

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.

Abstract

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 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.

Introduction

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 pores^2,3,4. The gasdermin pore provides an atypical secretion channel across cellular membranes, resulting in downstream cell responses, including content release and ion influx^2,3,4. 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 formation^6,8. 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 rupture^6,9. 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 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 integrity^11,12. 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.

Protocol

1. Cell line and cell culture

1. 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 β-mercaptoethanol. Culture the cells at 37 °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.
2. 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.

2. Differentiation of THP-1 cells

1. 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.
2. 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.
3. Open the cell counter and the counting software. Pipette 20 µL 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.
4. 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 °C and 5% CO₂ before starting further treatment.

3. Treatment of THP-1cells

1. 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 µg/mL. Prepare a stock solution of Na₂ATP with a concentration of 500 mM (dissolve 0.3026 g of ATP in 1 mL of PBS).
2. 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 °C + 5% CO₂ before proceeding with further treatment.
3. 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 °C + 5% CO₂ before proceeding.

4. Flow cytometry analysis (Method 1)

1. Preparation of experimental samples
   1. 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.
   2. After treatment, aspirate all the supernatant from the wells and wash with PBS 2x. Add 500 µL of 0.05% Trypsin (non-EDTA) to each well, and incubate in the incubator for 30 s.
   3. 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.
   4. Add 1 mL of PBS to resuspend cells. Place tubes with cells in a water bath at 37 °C for 3 min. Centrifuge all samples at 300 x g for 3 min at room temperature.
   5. Dilute 200 µL of 5x binding buffer with 800 µL of distilled water to 1x binding buffer. Add 100 µL of 1x binding buffer to resuspend cells and transfer the cells into the 5 mL tube.
   6. Add 5 µL of Annexin V-PE solution to one tube of control group cells for 10 min and 5 µL 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 µL of PBS to each tube and vortex gently to terminate incubation.
   7. Add 5 µL 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 µL of 7-AAD solution for 5 min at room temperature. Add 400 µL of PBS to each tube and vortex gently to terminate the incubation.
   8. Filter the cell suspension using a 35 µm 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.
2. Flow cytometry
   1. 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 > Clean Modes > De-gas Flow cell.
   2. 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.
   3. 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.
   4. 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.
   5. Click Next tube, run single staining controls of Annexin V and 7-AAD to adjust compensation. Run the remaining tubes and collect data.

5. SEM imaging (Method 2)

1. Preparation of experimental samples
   1. Seed and incubate cells in 6-well plates according to step 2 and treat according to step 3.
   2. After treatment, aspirate all the supernatant from the wells and wash 2x with PBS. Add 500 µL of 0.05% Trypsin (non-EDTA) to each well, and incubate in the incubator for 30 s.
   3. 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.
   4. Fix cells with 500 µL of electron microscope fixative (2.5% glutaric dialdehyde, 100 mM phosphorous salts) for 2 h at room temperature, followed by overnight at 4 °C.
   5. Prepare chromium alum solution: Add 1.0 g of gelatin to 100 mL of ultrapure water, 70 °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 °C and dry it in a 37 °C oven for later use.
      NOTE: The purpose is to prevent cell detachment during the experiment.
   6. Collect cells from fixed solution. Centrifuge at 300 x g for 3 min at room temperature. Add 100 µL 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.
   7. Add 500 μL of 1% osmic acid fixation for 1 hour. Aspirate all the supernatant and wash 3x with PBS for 5 min each time.
   8. 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 be kept in 100% EtOH for several days at 4 °C but it should dry as soon as possible to avoid excessive dehydration.
   9. Switch on the critical point dryer, open the CO₂ tank, cool the chamber to 10 °C. Quickly wrap the sample with filter paper when the chamber pressure is 0 psi and put it into the chamber.
   10. 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.
   11. Set the temperature to 35 °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.
   12. 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.
2. Imaging
   1. 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.

Results

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 ...

Discussion

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...

Materials

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