Name: Studying Copper Nanoparticle-Induced Programmed Cell Death in Bacteria (Video) | JoVE Journal
Uploaded: 2025-05-16T13:30:00.000+00:00
Duration: 8 min 22 s
Description: Explore copper nanoparticle-induced programmed cell death in bacteria using ROS generation assays and cell modulators. This method reveals antimicrobial mechanisms in Escherichia coli, Staphylococcus aureus, and Acinetobacter baumannii.

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5 minutes

We are grateful for the support from the Core Facility Center, Tzu Chi University, Taiwan.

The reagents and the equipment used in this study are listed in the Table of Materials.
1. Preparation of copper nanoparticle
<ol>
	<li>Obtain commercial copper nanopowders (25 nm and 60-80 nm) from a commercial source.</li>
	<li>Use 1.0 mM sodium dodecyl sulfate (SDS) as a dispersant for two sizes of 1 mg/mL nanoparticles.</li>
	<li>Disperse the nanoparticles using an ultrasonic bath for at least 30 min at room temperature. The fully dispersed nanoparticles are then ready for use in subsequent experiments.</li>
</ol>
2. Preparation of bacteria
<ol>
	<li>Obtain E. coli (Migula) Castellani and Chalmers strain 25922 and A. baumannii Bouvet and Grimont strain from the American Type Culture Collection. Obtain S. aureus from the Bioresource Collection and Research Center.</li>
	<li>Culture the bacteria in Luria-Bertani (LB) broth under aerobic conditions at 37 &#176;C.</li>
	<li>Dilute the bacterial cultures in LB medium to an optical density at 600 nm (OD600) of approximately 0.5.</li>
</ol>
3. Cell viability assessment
<ol>
	<li>Colony assay
	<ol>
		<li>Use stock CuNP solutions (1 mg/mL) to prepare various concentrations of two sizes of CuNPs, including 0 &#181;g/mL, 1 &#181;g/mL, 5 &#181;g/mL, 10 &#181;g/mL, 50 &#181;g/mL, and 100 &#181;g/mL.</li>
		<li>Split the bacterial cultures prepared in step 2.3 into microcentrifuge tubes and centrifuge at 3300 &#215; g for 10 min at room temperature.</li>
		<li>Retain the bacterial pellets and add different concentrations of two sizes of CuNPs, respectively, with gentle pipetting.</li>
		<li>Treat bacterial pellets with PBS and 70% alcohol as the negative and positive controls, respectively.</li>
		<li>Incubate all treated bacteria with shaking at 200 rpm at 37 &#176;C for 24 h.</li>
		<li>After incubation, wash all treated bacteria with PBS and spread them on LB agar plates. Place the plates in a 37 &#176;C incubator for 24 h.</li>
		<li>Count the colony numbers in each treatment group the next day and perform statistical analysis. It is recommended to conduct this in triplicate for statistical accuracy.</li>
	</ol>
	</li>
	<li>Bactericidal mechanism study
	<ol>
		<li>Prepare bacteria as described in step 3.1.2 and treat them with either 5 &#181;M of SBI-0206965 (SBI) for 2 h, 0.5 &#181;M of necrosulfonamide (NSA) for 1 h, 100 nM of wortmannin (Wort) for 30 min, or 100 nM of Z-VAD-FMK (Z-VAD) for 30 min.</li>
		<li>Co-treat the bacteria with different concentrations of two sizes of CuNP solutions, as described in step 3.1.1, in the presence or absence of 5 &#181;M of SBI, 0.5 &#181;M of NSA, 100 nM of Wort, and 100 nM of Z-VAD.</li>
		<li>Centrifuge the bacteria after modulator treatments (step 3.2.1) and remove the supernatants.</li>
		<li>Resuspend the bacterial pellets in solutions prepared in step 3.2.2 and incubate them with shaking at 200 rpm at 37 &#176;C for 24 h.</li>
		<li>Treat the bacteria with 70% ethanol and PBS as the positive and negative controls, respectively. Use a solution without CuNPs as the CuNP blank control (0 &#181;g/mL; mock) under the same inhibitor conditions for each group. Incubate all samples for an additional 24 h.</li>
		<li>After incubation, add cell viability reagent to the cultures at a 1:10 volume ratio. Incubate the cultures for another 2 h with shaking at 37 &#176;C.</li>
		<li>Centrifuge the cultures (step 3.1.2) after the 2 h incubation. Transfer the fluorescent supernatants to 96-well plates. Measure fluorescence using an excitation wavelength of 560 nm and an emission wavelength of 590 nm with a microplate reader.</li>
		<li>Dilute the remaining supernatant to 10-5&#160;and 10-4 and spread it onto LB agar plates for culturing.</li>
		<li>Count single colonies the following day.</li>
	</ol>
	</li>
</ol>
4. Detection of reactive oxygen species
<ol>
	<li>Prepare bacterial cultures as described in step 2.3 and split them into microcentrifuge tubes.</li>
	<li>Treat the bacteria with various stress conditions as ROS-inducing positive control groups (data not shown in the results). The treatments are described in steps 4.2.1-4.2.4.
	<ol>
		<li>Expose the bacteria to 405 nm UV light for 3 h. Incubate bacteria at 45 &#176;C for 2 h.</li>
		<li>Next, incubate bacteria at 4 &#176;C for 2 h.</li>
		<li>Treat bacteria with 3% H2O2 for 30 min.</li>
		<li>Maintain the bacteria at 37 &#176;C in LB broth as the negative control.</li>
	</ol>
	</li>
	<li>Prepare various concentrations of CuNPs as described in step 3.1.1, and treat the bacteria with 20 nm or 60 nm CuNPs at concentrations of 1 &#181;g/mL, 5 &#181;g/mL, 10 &#181;g/mL, and 100 &#181;g/mL for 24 h.</li>
	<li>Wash the incubated bacteria twice with PBS to remove any remaining nanoparticles.</li>
	<li>Prepare 2&#8242;,7&#8242;-dichlorodihydrofluorescein diacetate (H2DCFDA) dye in PBS at a final concentration of 5 &#181;M.</li>
	<li>Resuspend the bacterial pellets in 5 &#181;M of H2DCFDA and measure the fluorescence intensity at 520/30 nm emission using a flow cytometer. 
	NOTE: The intensity of FL1 green fluorescence correlates with the ROS level in the treated culture. It is recommended to perform this in triplicate for statistical accuracy.</li>
</ol>

<ol><li>Anees Ahmad, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bactericidal+activity+of+silver+nanoparticles%3A+A+mechanistic+review%5BTitle%5D)+AND+%22Mater+Sci+Energy+Technol%22%5BJournal%5D)">Bactericidal activity of silver nanoparticles: A mechanistic review.</a> Mater Sci Energy Technol. 3, 756-769 (2020).</li>
<li>Zhang, Y., Shareena Dasari, T. P., Deng, H., Yu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antimicrobial+activity+of+gold+nanoparticles+and+ionic+gold%5BTitle%5D)+AND+%22J+Environ+Sci+Health+C+Environ+Carcinog+Ecotoxicol+Rev%22%5BJournal%5D)">Antimicrobial activity of gold nanoparticles and ionic gold.</a> J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 33 (3), 286-327 (2015).</li>
<li>Solioz, M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSolioz%2C+M+%22Copper+and+Bacteria%3A+Evolution%2C+Homeostasis+and+ToxicitySpringerBriefs+in+Molecular+Science%22">Copper and Bacteria: Evolution, Homeostasis and ToxicitySpringerBriefs in Molecular Science</a>. Ch 2, Springer Cham. 11-19 (2018).</li>
<li>Ma, X., Zhou, S., Xu, X., Du, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Copper-containing+nanoparticles%3A+Mechanism+of+antimicrobial+effect+and+application+in+dentistry-a+narrative+review%5BTitle%5D)+AND+%22Front+Surg%22%5BJournal%5D)">Copper-containing nanoparticles: Mechanism of antimicrobial effect and application in dentistry-a narrative review.</a> Front Surg. 9, 905892(2022).</li>
<li>Draviana, H. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Size+and+charge+effects+of+metal+nanoclusters+on+antibacterial+mechanisms%5BTitle%5D)+AND+%22J+Nanobiotechnology%22%5BJournal%5D)">Size and charge effects of metal nanoclusters on antibacterial mechanisms.</a> J Nanobiotechnology. 21 (1), 428(2023).</li>
<li>Hasanuzzaman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+ROS+metabolism+in+plants+under+environmental+stress%3A+A+review+of+recent+experimental+evidence%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Regulation of ROS metabolism in plants under environmental stress: A review of recent experimental evidence.</a> Int J Mol Sci. 21 (22), ijms21228695(2020).</li>
<li>Lai, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+size+and+concentration+of+copper+nanoparticles+on+the+antimicrobial+activity+in+Escherichia+coli+through+multiple+mechanisms%5BTitle%5D)+AND+%22Nanomaterials+%28Basel%29%22%5BJournal%5D)">Effect of size and concentration of copper nanoparticles on the antimicrobial activity in Escherichia coli through multiple mechanisms.</a> Nanomaterials (Basel). 12 (21), 12213715(2022).</li>
<li>Sai, D. L., Lee, J., Nguyen, D. L., Kim, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tailoring+photosensitive+ROS+for+advanced+photodynamic+therapy%5BTitle%5D)+AND+%22Exp+Mol+Med%22%5BJournal%5D)">Tailoring photosensitive ROS for advanced photodynamic therapy.</a> Exp Mol Med. 53 (4), 495-504 (2021).</li>
<li>Peeters, S. H., de Jonge, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((For+the+greater+good%3A+Programmed+cell+death+in+bacterial+communities%5BTitle%5D)+AND+%22Microbiol+Res%22%5BJournal%5D)">For the greater good: Programmed cell death in bacterial communities.</a> Microbiol Res. 207, 161-169 (2018).</li>
<li>Allocati, N., Masulli, M., Di Ilio, C., De Laurenzi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Die+for+the+community%3A+An+overview+of+programmed+cell+death+in+bacteria%5BTitle%5D)+AND+%22Cell+Death+Dis%22%5BJournal%5D)">Die for the community: An overview of programmed cell death in bacteria.</a> Cell Death Dis. 6 (1), e1609(2015).</li>
<li>Kolodkin-Gal, I., Sat, B., Keshet, A., Engelberg-Kulka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+communication+factor+EDF+and+the+toxin-antitoxin+module+mazEF+determine+the+mode+of+action+of+antibiotics%5BTitle%5D)+AND+%22PLoS+Biol%22%5BJournal%5D)">The communication factor EDF and the toxin-antitoxin module mazEF determine the mode of action of antibiotics.</a> PLoS Biol. 6 (12), e319(2008).</li>
<li>Lai, M. J., Huang, Y. W., Wijaya, J., Liu, B. R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLai%2C+MJ+%22Copper+Overview%22">Copper Overview</a>. Fernández, G. D. Ch 4, IntechOpen. (2024).</li>
<li>Macomber, L., Imlay, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+iron-sulfur+clusters+of+dehydratases+are+primary+intracellular+targets+of+copper+toxicity%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">The iron-sulfur clusters of dehydratases are primary intracellular targets of copper toxicity.</a> Proc Natl Acad Sci U S A. 106 (20), 8344-8349 (2009).</li>
<li>Ramos-Zúñiga, J., Bruna, N., Pérez-Donoso, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toxicity+mechanisms+of+copper+nanoparticles+and+copper+surfaces+on+bacterial+cells+and+viruses%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Toxicity mechanisms of copper nanoparticles and copper surfaces on bacterial cells and viruses.</a> Int J Mol Sci. 24 (13), ijms241310503(2023).</li>
<li>Ihara, M., Shichijo, K., Takeshita, S., Kudo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wortmannin%2C+a+specific+inhibitor+of+phosphatidylinositol-3-kinase%2C+induces+accumulation+of+DNA+double-strand+breaks%5BTitle%5D)+AND+%22J+Radiat+Res%22%5BJournal%5D)">Wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, induces accumulation of DNA double-strand breaks.</a> J Radiat Res. 61 (2), 171-176 (2020).</li>
<li>Yang, Y. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+and+interpretation+of+current+autophagy+inhibitors+and+activators%5BTitle%5D)+AND+%22Acta+Pharmacol+Sin%22%5BJournal%5D)">Application and interpretation of current autophagy inhibitors and activators.</a> Acta Pharmacol Sin. 34 (5), 625-635 (2013).</li>
<li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+caspase+inhibitor+Z-VAD-FMK+alleviates+endotoxic+shock+via+inducing+macrophages+necroptosis+and+promoting+MDSCs-mediated+inhibition+of+macrophages+activation%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">The caspase inhibitor Z-VAD-FMK alleviates endotoxic shock via inducing macrophages necroptosis and promoting MDSCs-mediated inhibition of macrophages activation.</a> Front Immunol. 10, 1824(2019).</li>
<li>Solioz, M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSolioz%2C+M+%22Copper+and+Bacteria%3A+Evolution%2C+Homeostasis+and+ToxicitySpringerBriefs+in+Molecular+Science%22">Copper and Bacteria: Evolution, Homeostasis and ToxicitySpringerBriefs in Molecular Science</a>. Chapter 3, Springer. Cham. 21-48 (2018).</li>
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The author declares no conflict of interest, financial or otherwise.

This study investigated the antimicrobial effects and mechanisms of CuNPs at two sizes and various concentrations against E. coli, S. aureus, and A. baumannii. Using the established protocols, it was observed that CuNP-induced bactericidal effects involve oxidative stress and potential PCD activation. However, the interplay between metal homeostasis and bacterial stress responses remains largely unexplored. Previous studies have identified bacterial resistance strategies, such as copper efflux ...

Drug-resistant bacteria are a serious cause of concern in medicine. Their rapid emergence has reduced the efficacy of conventional antibiotics, resulting in more clinical complications. They pose a major threat to public health and create an urgent need for new antimicrobial agents. One avenue of research is nanomaterials. Nanomaterials possess unique physicochemical properties that allow them to interact with microbes in ways that compromise their viability. For instance, silver nanoparticles (AgNPs) induce oxidative stress in bacteria, resulting in protein dysfunction, membrane disruption, DNA damage, and ultimately cell death1. Gold nanoparticles (AuNPs), on the other hand, are known for their antifungal properties and can enhance the bactericidal effect of antibiotics by serving as carriers2.
Additionally, copper nanoparticles (CuNPs) have also attracted considerable attention due to their potent antimicrobial effect and low production cost. Studies suggest that CuNPs exhibit broad-spectrum bactericidal activity by disruption of enzymatic activity and the generation of reactive oxygen species (ROS)3. The positive charge of CuNPs facilitates their penetration into the bacteria, enhancing their cellular uptake4. This mechanism makes CuNPs a promising option for surface coating, such as on implants, to prevent infections3. One interesting finding, however, is that the bactericidal effect of CuNPs appears to be size-dependent. Some studies have found that smaller CuNPs exhibit higher antibacterial activity, probably due to their superior surface area-to-volume ratio5.
ROS generation causes widespread damage to cells and bacteria, including lipid peroxidation, protein dysfunction, DNA fragmentation, and inhibition of gluconeogenesis/glycogenolysis, and is involved in necrosis or programmed cell death (PCD)6,7,8. Recent studies have revealed that PCD systems exist in bacteria, with action modes and effectors similar to those in eukaryotic systems9. Bacterial communities can induce PCD in response to stress, including oxidative stress, through a toxin-antitoxin (TA) system10. In simple terms, the toxin-antitoxin system consists of toxins that can disrupt essential cellular processes and antitoxins that can form stable complexes with the toxins to inhibit their toxicity under normal growth conditions. Most bacteria and archaea contain TA loci in their genomes, often present in multiple copies of extrachromosomal and chromosomal DNA. There are several types of TA systems, with type II TA (known as MazE/MazF module) being of particular interest. Under stress conditions, antitoxins are degraded, allowing toxins to inhibit their cellular targets. In E. coli and S. aureus, the toxin MazF is activated in response to stress conditions such as oxidative stress, high temperature, and amino acid starvation. Consequently, the expression of the antitoxin MazE is reduced, releasing the toxin MazF10. Studies have found that MazF enables the synthesis of proteins that allow a small sub-population to survive under adverse conditions, while most of the population undergoes mazEF-mediated cell death. This cell death can be either ROS-dependent, where ROS induces transcriptional or translational inhibition, or ROS-independent, where DNA damage triggers the death pathways11.
This study explores the mechanisms by which CuNPs induce bacterial death. Rather than focusing solely on the TA system, four PCD modulators, previously used in our research7,12, were employed to investigate potential PCD pathways in bacteria.
By examining the bactericidal effects of CuNPs of two different sizes (20 and 60 nm) at varying concentrations, and utilizing methods such as colony assays, ROS detection, and PCD modulators (SBI, Z-VAD, NSA, and Wortmannin), this research highlights that PCD is not exclusive to multicellular organisms but also occurs in bacterial communities under stress. By providing detailed protocols, this work aims to enable researchers to evaluate CuNP efficacy and bactericidal mechanisms in their own systems. Furthermore, these findings advance the understanding of bacterial PCD and support the development of CuNP-based therapies to combat antibiotic-resistant bacteria.

<table><tbody><tr><td>&lt;em&gt;Acinetobacter baumannii&lt;/em&gt; Bouvet and Grimont strain&amp;nbsp;</td><td>American Type Culture Collection (ATCC), Manassas, VA, USA</td><td>17978</td><td>Bacteria for CuNP toxocity experiment</td></tr><tr><td>Bio-Rad iMark Microplate Reader</td><td>Bio-Rad Laboratories, Hercules, CA, USA</td><td>168-1130</td><td>Used to measure absorbance in bacterial viability assays.</td></tr><tr><td>cell-permeant 2&amp;rsquo;,7&amp;rsquo;-dichlorodihydrofluorescein diacetate&amp;nbsp; (H2DCFDA)</td><td>Sigma-Aldrich, Saint Louis, MO, USA</td><td>D6883</td><td>Used for detecting reactive oxygen species (ROS) in treated bacterial cells.</td></tr><tr><td>Copper nanoparticles (CuNPs) 25 nm</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>774081</td><td>Used to prepare CuNP stock solution</td></tr><tr><td>Copper nanoparticles (CuNPs) 60-80 nm</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>774103</td><td>Used to prepare CuNP stock solution</td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; (Migula) Castellani and Chalmers</td><td>American Type Culture Collection (ATCC), Manassas, VA, USA</td><td>25922</td><td>Bacteria for CuNP toxocity experiment</td></tr><tr><td>Gallios flow cytometer</td><td>Beckman Coulter, Brea, CA, USA</td><td></td><td>Used for flow cytometric analysis in multiple experiments, including reactive oxygen species detection.</td></tr><tr><td>LB agar</td><td>FocusBio, Miaoli, Taiwan</td><td>LBA500</td><td>Used for culturing bacteria</td></tr><tr><td>Luria-Bertani (LB) broth</td><td>Becton, Dickinson and Company, Sparks, MD, USA</td><td>244620</td><td>Used for culturing bacteria</td></tr><tr><td>Necrosulfonamide (NSA)</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>480073</td><td>Used as a modulator for pretreatment in bacterial death pathway studies.</td></tr><tr><td>PrestoBlue Cell Viability Reagent</td><td>Invitrogen, Carlsbad, CA, USA</td><td>P50200</td><td>Used for assessing cell viability via fluorescence.</td></tr><tr><td>SBI-0206965 (SBI)</td><td>BioVision, Milpitas, CA, USA</td><td>9580</td><td>Used as a modulator for pretreatment in bacterial death pathway studies.</td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>L4509</td><td>Used as a dispersant for copper nanoparticles to reduce aggregation.</td></tr><tr><td>&lt;em&gt;Staphylococcus aureus&lt;/em&gt;</td><td>American Type Culture Collection (ATCC), Manassas, VA, USA&lt;br/&gt; Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan</td><td>13567</td><td>Bacteria for CuNP toxocity experiment</td></tr><tr><td>Varioskan LUX multimode microplate reader</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>VLBLATGD2</td><td>Used for measuring fluorescence in cell viability assays</td></tr><tr><td>Wortmannin (Wort)</td><td>Abcam, MA, USA</td><td>ab120148</td><td>Used as a modulator for pretreatment in bacterial death pathway studies.</td></tr><tr><td>Z-VAD-FMK (Z-VAD)</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>V116</td><td>Used as a modulator for pretreatment in bacterial death pathway studies.</td></tr></tbody></table>

studying copper nanoparticle-induced programmed cell death in bacteria

Studying Copper Nanoparticle-Induced Programmed Cell Death in Bacteria

Recently, concerns over multidrug-resistant pathogens and incurable infections have increased due to the overuse and misuse of antibiotics. Nanomaterials, such as metallic and metallic oxide nanoparticles, have gained popularity in the biomedical field as potential new strategies to combat multidrug-resistant pathogens. This study investigated the use of copper nanoparticles (CuNPs) as a bactericide against three common hospital-acquired opportunistic pathogens-Escherichia coli (E. coli), Acinetobacter baumannii (A. baumannii), and Staphylococcus aureus (S. aureus)-which are increasingly developing drug resistance. Detailed protocols are presented for synthesizing CuNPs of two sizes (20 nm and 60 nm) and evaluating their bactericidal efficacy through colony assays. The mechanisms of antimicrobial action underlying CuNPs were explored by assessing changes in reactive oxygen species production. Additionally, four modulators that inhibit human protein functions were applied to study the potential involvement of programmed cell death (PCD) pathways in bacterial killing. Through this approach, the potential emergence of copper-resistant strains is suggested, building on research into copper homeostasis proteins, including copper-dependent transcriptional regulators. These findings provide a comprehensive methodology for studying the bactericidal effects of CuNPs and their potential role in addressing antibiotic resistance.

Antimicrobial activities of two-size CuNPs in three pathogens 
Three opportunistic pathogens (E. coli, S. aureus, and A. baumannii) were used to test the bactericidal activities of CuNPs. The bacteria were treated with 0 µg/mL, 1 µg/mL, 5 µg/mL, 10 µg/mL, 50 µg/mL, and 100 µg/mL of 20 nm or 60 nm CuNPs, and the bactericidal activities were determined using the minimum bactericidal concentration (MBC) derived from colony counts. Our results showed positive bactericidal effect...

Copper nanoparticles act as antimicrobial agents by generating reactive oxygen species. Here, procedures are presented demonstrating that copper nanoparticles are effective against three clinically relevant pathogens and that certain programmed cell death pathways are involved in this bactericidal process.

Recently, concerns over multidrug-resistant pathogens and incurable infections have increased due to the overuse and misuse of antibiotics. Nanomaterials, ...

studying-copper-nanoparticle-induced-programmed-cell-death-in-bacteria

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Fabrication of Antibacterial Graphene Oxide/Copper Nanocomposites

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

Explore copper nanoparticle-induced programmed cell death in bacteria using ROS generation assays and cell modulators. This method reveals antimicrobial mechanisms in Escherichia coli, Staphylococcus aureus, and Acinetobacter baumannii.

Studying Copper Nanoparticle-Induced Programmed Cell Death in Bacteria

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