Optimized Scratch Assay for &lt;em&gt;In Vitro&lt;/em&gt; Testing of Cell Migration with an Automated Optical Camera

Mouritzen, Michelle Vang; Jenssen, Håvard

doi:10.3791/57691-v

Cited by 13 publications

(12 citation statements)

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“…Two methods are generally used to evaluate the cell migration of HaCaT, namely wound scratch assay and transwell cell migration assay. The wound scratch assay is the most common in vitro method to test compounds for their migratory properties due to its simplicity and cost-effective procedure [ 107 ]. Additionally, the scratch assay is comparable to the in vivo wound healing process because it involves the removal of cells using a pipette tip, which wounds the monolayer and causes destroyed and activated cells [ 108 ].…”

Section: Resultsmentioning

confidence: 99%

“…In addition, HaCaT cells have strong cell-to-cell interaction and are facilitated by Ca 2+ -dependent interactions at the desmosome junction. Hence, prior to the scratch, washing with PBS is an essential step to weaken the cell-to-cell adhesion of HaCaT for easy removal, eliminating one of the disadvantages of performing manual scratches, as the cells tend to accumulate across the edge of the scratch due to this strong interaction [ 107 ].…”

Section: Resultsmentioning

confidence: 99%

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Antibacterial, Anti-Biofilm and Pro-Migratory Effects of Double Layered Hydrogels Packaged with Lactoferrin-DsiRNA-Silver Nanoparticles for Chronic Wound Therapy

Fathil

Katas

2023

Pharmaceutics

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Antimicrobial resistance and biofilm formation in diabetic foot infections worsened during the COVID-19 pandemic, resulting in more severe infections and increased amputations. Therefore, this study aimed to develop a dressing that could effectively aid in the wound healing process and prevent bacterial infections by exerting both antibacterial and anti-biofilm effects. Silver nanoparticles (AgNPs) and lactoferrin (LTF) have been investigated as alternative antimicrobial and anti-biofilm agents, respectively, while dicer-substrate short interfering RNA (DsiRNA) has also been studied for its wound healing effect in diabetic wounds. In this study, AgNPs were complexed with LTF and DsiRNA via simple complexation before packaging in gelatin hydrogels. The formed hydrogels exhibited 1668% maximum swellability, with a 46.67 ± 10.33 µm average pore size. The hydrogels demonstrated positive antibacterial and anti-biofilm effects toward the selected Gram-positive and Gram-negative bacteria. The hydrogel containing AgLTF at 125 µg/mL was also non-cytotoxic on HaCaT cells for up to 72 h of incubation. The hydrogels containing DsiRNA and LTF demonstrated superior pro-migratory effects compared to the control group. In conclusion, the AgLTF-DsiRNA-loaded hydrogel possessed antibacterial, anti-biofilm, and pro-migratory activities. These findings provide a further understanding and knowledge on forming multipronged AgNPs consisting of DsiRNA and LTF for chronic wound therapy.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Antibacterial, Anti-Biofilm and Pro-Migratory Effects of Double Layered Hydrogels Packaged with Lactoferrin-DsiRNA-Silver Nanoparticles for Chronic Wound Therapy

Fathil

Katas

2023

Pharmaceutics

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“…Invasion and migration experiments in Transwell chambers and wound healing assay were undertaken to evaluate cells' capacity for metastasis. The wound healing experiment was executed following the routine workflow 18 . Cells in dishes were pretreated with the proliferation inhibitor mitomycin C (0.5 μM) for 2 h, which effectively inhibited HCC cell proliferation without significantly increasing cytotoxicity.…”

Section: Methodsmentioning

confidence: 99%

“…The wound healing experiment was executed following the routine workflow. 18 Cells in dishes were pretreated with the proliferation inhibitor mitomycin C (0.5 μM) for 2 h, which effectively inhibited HCC cell proliferation without significantly increasing cytotoxicity. Wound healing was observed and photographs were taken 48 h after scratching.…”

Section: Cell Migration and Invasion Assaysmentioning

confidence: 99%

Downregulated mitochondrial transcription factor A enhances mycoplasma infection to promote the metastasis of hepatocellular carcinoma

Wang

Hong

et al. 2023

Cancer Science

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Mycoplasma is widespread in various hosts and may cause various diseases in animals. Interestingly, the occurrence of mycoplasma infection was observed in many tumor types. However, the mechanism regulating its infection is far from clear. We unexpectedly found that the knockdown of mitochondrial transcription factor A (TFAM) remarkably enhanced mycoplasma infection in hepatocellular carcinoma (HCC) cells. More importantly, we found that mycoplasma infection facilitated by TFAM knockdown significantly promoted HCC cell metastasis. Mycoplasma infection was further found to be positively correlated with poor prognosis in patients with HCC. Mechanistically, the decreased TFAM expression upregulated the transcription factor Sp1 to increase the expression level of Annexin A2 (ANXA2), which was reported to interact with membrane protein of mycoplasma. Moreover, we found that mycoplasma infection enhanced by the TFAM downregulation promoted HCC migration and invasion by activating the nuclear factor‐κB signaling pathway. The downregulation of TFAM enhanced mycoplasma infection in HCC cells and promoted HCC cell metastasis. Our study contributes to the understanding of the pathological role of mycoplasma infection and provides supporting evidence that targeting TFAM could be a potential strategy for the treatment of HCC with mycoplasma infection.

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“…The gold standard in migration assay involves creating a scratch on a petri dish using a sterile pipette ( Michalczyk et al, 2018 ), which disrupts the cell monolayer and generates an empty space devoid of cells ( Mouritzen and Jenssen, 2018 ). Additionally, gold nanoparticle (GNP) coating aids in the quantification of cell movements, including nanoparticles functionalized with growth factors ( Leu et al, 2012) .…”

Section: Introductionmentioning

confidence: 99%

Tradeoffs in ATP metabolisms via hypoxic gradient migration assays

Orabi,

Duan,

Zhou

et al. 2024

Preprint

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Migration and scratch assays are helpful tools to investigate wound healing and tissue regeneration processes, especially under disease conditions such as diabetes. However, traditional migration (injury-free) assays and scratch (with injury) assays are limited in their control over cellular environments and provide only simplified read-outs of their results. On the other hand, microfluidic-based cell assays offer a distinct advantage in their integration and scalability for multiple modalities and concentrations in a single device. Additionally, in situ stimulation and detection helps to avoid variabilities between individual bioassays. To realize an enhanced, smarter migration assay, we leveraged our multilayered oxygen gradient (1-16%) to study HaCaT migrations in diabetic conditions with spatial and metabolic read-outs. An analysis of the spatial migration over time observed a new dynamic between hypoxia (at 4.16-9.14% O2) and hyperglycemia. Furthermore, in situ adenosine triphosphate (ATP) and reactive oxygen species (ROS) responses suggest that this dynamic represents a switch between stationary versus motile modes of metabolism. Thus, elevated glucose and hypoxia are synergistic triggers of this switch under disease conditions. These findings illustrate the benefits of spatial microfluidics for modeling complex diseases such as hypoxia and diabetes, where multimodal measurements provide a more deterministic view of the underlying processes.

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Optimized Scratch Assay for <em>In Vitro</em> Testing of Cell Migration with an Automated Optical Camera

Cited by 13 publications

References 0 publications

Antibacterial, Anti-Biofilm and Pro-Migratory Effects of Double Layered Hydrogels Packaged with Lactoferrin-DsiRNA-Silver Nanoparticles for Chronic Wound Therapy

Antibacterial, Anti-Biofilm and Pro-Migratory Effects of Double Layered Hydrogels Packaged with Lactoferrin-DsiRNA-Silver Nanoparticles for Chronic Wound Therapy

Downregulated mitochondrial transcription factor A enhances mycoplasma infection to promote the metastasis of hepatocellular carcinoma

Tradeoffs in ATP metabolisms via hypoxic gradient migration assays

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