Fluorescent Labeling Method Research Articles

FLUID: a fluorescence-friendly lipid-compatible ultrafast clearing method.

Many clearing methods achieve high transparency by removing lipid components from tissues, which damages microstructure and limits their application in lipid research. As for methods which preserve lipid, it is difficult to balance transparency, fluorescence preservation and clearing speed. In this study, we propose a rapid water-based clearing method that is fluorescence-friendly and preserves lipid components. FLUID allows for preservation of endogenous fluorescence over 60 days. It shows negligible tissue distortion and is compatible with various types of fluorescent labeling and tissue staining methods. High quality imaging of human brain tissue and compatibility with pathological staining demonstrated the potential of our method for three-dimensional (3D) biopsy and clinical pathological diagnosis.

Di-caffeoylquinic acid: a potential inhibitor for amyloid-beta aggregation.

Alzheimer's disease (AD) remains a challenging neurodegenerative disorder with limited therapeutic success. Traditional Chinese Medicine (TCM), as a promising new source for AD, still requires further exploration to understand its complex components and mechanisms. Here, focused on addressing Aβ (1-40) aggregation, a hallmark of AD pathology, we employed a Thioflavin T fluorescence labeling method for screening the active molecular library of TCM which we established. Among the eight identified, 1,3-di-caffeoylquinic acid emerged as the most promising, exhibiting a robust binding affinity with a KD value of 26.7nM. This study delves into the molecular intricacies by utilizing advanced techniques, including two-dimensional (2D) 15N-1H heteronuclear single quantum coherence nuclear magnetic resonance (NMR) and molecular docking simulations. These analyses revealed that 1,3-di-caffeoylquinic acid disrupts Aβ (1-40) self-aggregation by interacting with specific phenolic hydroxyl and amino acid residues, particularly at Met-35 in Aβ (1-40). Furthermore, at the cellular level, the identified compounds, especially 1,3-di-caffeoylquinic acid, demonstrated low toxicity and exhibited therapeutic potential by regulating mitochondrial membrane potential, reducing cell apoptosis, and mitigating Aβ (1-40)-induced cellular damage. This study presents a targeted exploration of catechol compounds with implications for effective interventions in AD and sheds light on the intricate molecular mechanisms underlying Aβ (1-40) aggregation disruption.

A novel multiplex fluorescent-labeling method for the visualization of mixed-species biofilms in vitro.

In nature, bacteria usually exist as mixed-species biofilms, where they engage in a range of synergistic and antagonistic interactions that increase their resistance to environmental challenges. Biofilms are a major cause of persistent infections, and dispersal from initial foci can cause new infections at distal sites thus warranting further investigation. Studies of development and spatial interactions in mixed-species biofilms can be challenging due to difficulties in identifying the different bacterial species in situ. Here, we apply CellTrace dyes to studies of biofilm bacteria and present a novel application for multiplex labeling, allowing identification of different bacteria in mixed-species, in vitro biofilm models. Oral bacteria labeled with CellTrace dyes (far red, yellow, violet, and CFSE [green]) were used to create single- and mixed-species biofilms, which were analyzed with confocal spinning disk microscopy (CSDM). Biofilm supernatants were studied with flow cytometry (FC). Both Gram-positive and Gram-negative bacteria were well labeled and CSDM revealed biofilms with clear morphology and stable staining for up to 4 days. Analysis of CellTrace labeled cells in supernatants using FC showed differences in the biofilm dispersal between bacterial species. Multiplexing with different colored dyes allowed visualization of spatial relationships between bacteria in mixed-species biofilms and relative coverage by the different species was revealed through segmentation of the CSDM images. This novel application, thus, offers a powerful tool for studying structure and composition of mixed-species biofilms in vitro.IMPORTANCEAlthough most chronic infections are caused by mixed-species biofilms, much of our knowledge still comes from planktonic cultures of single bacterial species. Studies of formation and development of mixed-species biofilms are, therefore, required. This work describes a method applicable to labeling of bacteria for in vitro studies of biofilm structure and dispersal. Critically, labeled bacteria can be multiplexed for identification of different species in mixed-species biofilms using confocal spinning disk microscopy, facilitating investigation of biofilm development and spatial interactions under different environmental conditions. The study is an important step in increasing the tools available for such complex and challenging studies.

Simple Fluorescence Labeling Method Enables Detection of Intracellular Distribution and Expression Level of Retinoid X Receptors.

There is no straightforward method to visualize the intracellular distribution of nuclear receptors, such as retinoid X receptors (RXRs), which are trafficked between the cytosol and nucleus. Here, in order to develop a simple fluorescence labeling method for RXRs, we designed and synthesized compound 4, consisting of an RXR-selective antagonist, CBTF-EE (2), linked via an ether bond to the fluorophore nitrobenzoxadiazole (NBD). Compound 4 is nonfluorescent, but the ether bond (-O-NBD) reacts with biothiols such as cysteine and homocysteine to generate a thioether (-S-NBD), followed by intramolecular Smiles rearrangement with an amino group such as that of lysine to form a fluorescent secondary amine (-NH-NBD) adjacent to the binding site. Fluorescence microscopy of intact or RXR-overexpressing MCF-7 cells after incubation with 4 enabled us to visualize RXR expression as well as nuclear transfer of RXR induced by the agonist bexarotene (1).

Isolation and functional analysis of drought related PobZIP4 transcription factor in Paeonia ostii

Drought, as a natural disaster of force majeure, significantly disrupts the physiological balance of plants and crop yields. The basic leucine zipper (bZIP) transcription factors assume the pivotal role of paramount regulators in orchestrating drought response. In this study, a bZIP transcription factor PobZIP4 was isolated on the base of Paeonia ostii transcriptome, and its function in response to drought stress was verified. PobZIP4 had a 579 bp open reading frame, encoded 192 amino acids, and existed in a conserved structural domain typical of bZIP, and was highly expressed when P. ostii was stressed by drought. Moreover, the subcellular localization of PobZIP4 was observed with fluorescent protein labeling method and it was found that PobZIP4 localized in the Nicotiana benthamiana epidermal cell nucleus. Then, we silenced PobZIP4 in P. ostii using virus-induced gene silencing technology, and found that PobZIP4-silenced plants showed significantly less tolerant to drought stress than the negative control. The accumulation of reactive oxygen species was found to be heightened, while the photosynthetic capacity, proline content, and protective enzyme activities exhibited a significant decline upon silencing PobZIP4, which unequivocally demonstrated that PobZIP4 amplifies the drought tolerance of P. ostii. This study highlighted the positive role of PobZIP4 in mitigating drought stress in P. ostii, thereby establishing a molecular basis for enhancing the drought tolerance of P. ostii.

Real-Time Monitoring of a Nucleic Acid Amplification Reaction Using a Mass Sensor Based on a Quartz-Crystal Microbalance.

Nucleic acid amplification reactions such as polymerase chain reaction (PCR), which uses a DNA polymerase to amplify individual double-stranded DNA fragments, are a useful technique for visualizing the presence of specific genomes. Although the fluorescent labeling method is mainly used with DNA amplification, other detection methods should be considered for further improvements, such as miniaturization and cost reduction, of reaction-monitoring devices. In this study, the quartz-crystal microbalance (QCM) method, which can measure nanogram-order masses, was applied for the real-time detection of DNA fragments in a solution with nucleic acids. This was combined with an isothermal nucleic acid amplification reaction based on the recombinase polymerase amplification (RPA) method, which allowed DNA amplification at a constant temperature. When the DNA amplification reaction was initiated on a QCM sensor plate with an immobilized primer DNA strand, a significant increase in mass was observed compared to when the primer DNA was not immobilized. QCM was shown to be sufficiently sensitive for the in situ detection of amplified DNA fragments. Combining a portable QCM device and RPA offers a sensitive point-of-care method for detecting nucleic acids.

Carbon Dots with Spatially-Mediated-N/S-Co-Doping Enabling One-Year Stable Lubricant with Oil Leakage Detection Capability.

The dispersion stability of nano-lubricating additives is crucial for the shelf life of lubricant and its practical applications. Nitrogen-sulfur co-doped carbon dots (N,S@CDs) via a one-step hydrothermal method with nitropyrene and thiourea as raw materials are hereby presented. The N and S elements are selectively distributed throughout the entire carbon skeleton with a doping amount of 22.6 at%. The as-synthesized N,S@CDs exhibit excellent dispersion stability in PEG200 and maintain stability for over one year. The experiment results indicate that N,S@CDs significantly improve the anti-wear and friction reduction properties of PEG200, while the friction coefficientis reduced from 0.25 to 0.09 with 1.5 wt% N,S@CDs addition, and the wear volume, depth, and width are reduced by 68%, 52%, and 57%, respectively. The good lubrication performance is attributed to N,S@CDs excellent dispersion stability, enhanced filling and polishing effects, and complex tribochemical reactions caused by heteroatom doping to form a stable protective film on the worn surface. Furthermore, the as-prepared N,S@CDs exhibit intrinsic fluorescence intensity in PEG200 with the photoluminescence quantum yield (PLQY) of 12.5% and remain fluorescent stable during the long-term friction process, therefore the N,S@CDs have a potential application prospect in non-destructive detection of oil leakage via fluorescence labeling method.

New protein fluorescent labeling methods for carbonylation and S-acylation studies in plants

New protein fluorescent labeling methods for carbonylation and S-acylation studies in plants

Imaging HIV-1 Nuclear Import, Uncoating, and Proviral Transcription.

Live-cell imaging has become a powerful tool for dissecting the behavior of viral complexes during HIV-1 infection with high temporal and spatial resolution. Very few HIV-1 particles in a viral population are infectious and successfully complete replication (~1/50). Single-particle live-cell imaging enables the study of these rare infectious viral particles, which cannot be accomplished in biochemical assays that measure the average property of the entire viral population, most of which are not infectious. The timing and location of many events in the early stage of the HIV-1 life cycle, including nuclear import, uncoating, and integration, have only recently been elucidated. Live-cell imaging also provides a valuable approach to study interactions of viral and host factors in distinct cellular compartments and at specific stages of viral replication. Successful live-cell imaging experiments require careful consideration of the fluorescent labeling method used and avoid or minimize its potential impact on normal viral replication and produce misleading results. Ideally, it is beneficial to utilize multiple virus labeling strategies and compare the results to ensure that the virion labeling did not adversely influence the viral replication step that is under investigation. Another potential benefit of using different labeling strategies is that they can provide information about the state of the viral complexes. Here, we describe our methods that utilize multiple fluorescent protein labeling approaches to visualize and quantify important events in the HIV-1 life cycle, including docking HIV-1 particles with the nuclear envelope (NE) and their nuclear import, uncoating, and proviral transcription.

Vitamin D3 induces stem cell activation via Lgr5-Bmi1 expression and improving mouse colitis histology index.

Conventional therapy for inflammatory bowel disease using long-term anti-inflammatory drugs does not seem to provide optimal results. Adjuvant therapy using vitamin D3 is believed to have an essential role in repairing the colonic mucosa through the activation of colonic stem cells. The aim of this study was to demonstrate the effect of vitamin D3 in mucosal repair through stem cell activation, marked by leucin-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and B lymphoma Mo-MLV insertion region 1 (Bmi1) expression and decrease the mouse colitis histology index (MCHI) score. In this study, 50 Mus musculus strain BALB/c were divided into five groups: negative control group, colitis group, and colitis groups with vitamin D3 administration of 0.2 mcg, 0.4 mcg, and 0.6 mcg per 25 g body weight for seven days. Dextran sulfate sodium (DSS) 5% was used to induce colitis. Lgr5-Bmi1 expression was measured using immunodoublestain fluorescent labeling method. Our data suggested that administration of vitamin D3 significantly increased expression of Lgr5-Bmi1 in the colonic mucosa. The colitis group treated with the highest dose of vitamin D3 (0.6 mcg/25 gram) showed the lowest MCHI score (3.60±0.64) while the lowest dose of vitamin D3 had the highest MCHI score (12.60±1.47). In conclusion, by stimulating stem cells, vitamin D3 administration stimulates mucosal regeneration, as demonstrated by upregulated expression of Lgr5-Bmi-1.

Danlou Recipe promotes cholesterol efflux in macrophages RAW264.7 and reverses cholesterol transport in mice with hyperlipidemia induced by P407

IntroductionLiver X Receptor (LXR) agonists could attenuate the development of atherosclerosis but bring excess lipid accumulation in the liver. Danlou Recipe was believed to be a benefit for improving the lipid profile. Thus, it is unclear whether Danlou Recipe could attenuate hyperlipidemia without excess lipid accumulated in the liver of mice. This study aimed to clarify if Danlou Recipe could alleviate the progression of hyperlipidemia in mice without extra lipids accumulated in the liver.MethodsMale murine macrophage RAW264.7 cells and murine peritoneal macrophages were used for the in vitro experiments. Cellular cholesterol efflux was determined using the fluorescent cholesterol labeling method. Those genes involved in lipid metabolism were evaluated by qRT‐PCR and western blotting respectively. In vivo, a mouse model of hyperlipidemia induced by P407 was used to figure out the effect of Danlou Recipe on reverse cholesterol transport (RCT) and hyperlipidemia. Ethanol extract of Danlou tablet (EEDL) was prepared by extracting the whole powder of Danlou Prescription from ethanol, and the chemical composition was analyzed by ultra-performance liquid chromatography (UPLC).ResultsEEDL inhibits the formation of RAW264.7 macrophage-derived foam cells, and promotes ABCA1/apoA1 conducted cholesterol efflux in RAW264.7 macrophages and mouse peritoneal macrophages. In the P407-induced hyperlipidemia mouse model, oral administration of EEDL can promote RCT in vivo and improve fatty liver induced by a high-fat diet. Consistent with the findings in vitro, EEDL promotes RCT by upregulating the LXR activities.ConclusionOur results demonstrate that EEDL has the potential for targeting RCT/LXR in the treatment of lipid metabolism disorders to be developed as a safe and effective therapy.

Metabolic labelling of a subpopulation of small extracellular vesicles using a fluorescent palmitic acid analogue.

Exosomes are among the most puzzling vehicles of intercellular communication, but several crucial aspects of their biogenesis remain elusive, primarily due to the difficulty in purifying vesicles with similar sizes and densities. Here we report an effective methodology for labelling small extracellular vesicles (sEV) using Bodipy FL C16, a fluorescent palmitic acid analogue. In this study, we present compelling evidence that the fluorescent sEV population derived from Bodipy C16-labelled cells represents a discrete subpopulation of small exosomes following an intracellular pathway. Rapid cellular uptake and metabolism of Bodipy C16 resulted in the incorporation of fluorescent phospholipids into intracellular organelles specifically excluding the plasma membrane and ultimately becoming part of the exosomal membrane. Importantly, our fluorescence labelling method facilitated accurate quantification and characterization of exosomes, overcoming the limitations of nonspecific dye incorporation into heterogeneous vesicle populations. The characterization of Bodipy-labelled exosomes reveals their enrichment in tetraspanin markers, particularly CD63 and CD81, and in minor proportion CD9. Moreover, we employed nanoFACS sorting and electron microscopy to confirm the exosomal nature of Bodipy-labelled vesicles. This innovative metabolic labelling approach, based on the fate of a fatty acid, offers new avenues for investigating exosome biogenesis and functional properties in various physiological and pathological contexts.

A Protocol for Activated Bioorthogonal Fluorescence Labeling and Imaging of 4‐Hydroxyphenylpyruvate Dioxygenase in Plants

Abstract4‐Hydroxyphenylpyruvate dioxygenase (HPPD) plays a crucial role in the synthesis of nutrients needed to maintain optimal plant growth. Its level is closely linked to the extent of abiotic stress experienced by plants. Moreover, it is also the target of commercial herbicides. Therefore, labeling of HPPD in plants not only enables visualization of its tissue distribution and cellular uptake, it also facilitates assessment of abiotic stress of plants and provides information needed for the development of effective environmentally friendly herbicides. In this study, we created a method for fluorescence labeling of HPPD that avoids interference with the normal growth of plants. In this strategy, a perylene‐linked dibenzyl‐cyclooctyne undergoes strain‐promoted azide‐alkyne cycloaddition with an azide‐containing HPPD ligand. The activation‐based labeling process results in a significant emission enhancement caused by the change in the fluorescent forms from an excimer to a monomer. Notably, this activated bioorthogonal strategy is applicable to visualizing HPPD in Arabidopsis thaliana, and assessing its response to multiple abiotic stresses. Also, it can be employed to monitor in vivo levels and locations of HPPD in crops. Consequently, the labeling strategy will be a significant tool in investigations of HPPD‐related abiotic stress mechanisms, discovering novel herbicides, and uncovering unknown biological functions.

A Protocol for Activated Bioorthogonal Fluorescence Labeling and Imaging of 4-Hydroxyphenylpyruvate Dioxygenase in Plants.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) plays a crucial role in the synthesis of nutrients needed to maintain optimal plant growth. Its level is closely linked to the extent of abiotic stress experienced by plants. Moreover, it is also the target of commercial herbicides. Therefore, labeling of HPPD in plants not only enables visualization of its tissue distribution and cellular uptake, it also facilitates assessment of abiotic stress of plants and provides information needed for the development of effective environmentally friendly herbicides. In this study, we created a method for fluorescence labeling of HPPD that avoids interference with the normal growth of plants. In this strategy, a perylene-linked dibenzyl-cyclooctyne undergoes strain-promoted azide-alkyne cycloaddition with an azide-containing HPPD ligand. The activation-based labeling process results in a significant emission enhancement caused by the change in the fluorescent forms from an excimer to a monomer. Notably, this activated bioorthogonal strategy is applicable to visualizing HPPD in Arabidopsis thaliana, and assessing its response to multiple abiotic stresses. Also, it can be employed to monitor in vivo levels and locations of HPPD in crops. Consequently, the labeling strategy will be a significant tool in investigations of HPPD-related abiotic stress mechanisms, discovering novel herbicides, and uncovering unknown biological functions.

Efficient and sustainable bleaching of cotton/spandex integrating UVA radiation and peroxide activator: A study focus on the degradation mechanism of natural pigments in fibres

Establishing an energy/water-saving bleaching process is highly expected by textile plants to reduce industrial carbon emission, wastewater discharge and general cost. Therefore, an efficient UVA-assisted N-[4-(triethylammoniomethyl)-benzoyl]- caprolactam-chloride (TBCC) -activated hydrogen peroxide (H2O2) bleaching strategy at ambient temperature for cotton/spandex (C/S) fabric was developed in this study. Results reveal that 7.4 g/L of TBCC-activated H2O2 (30%) achieves a slightly higher whiteness index (WI) on C/S fabrics than those only treated with 60 g/L H2O2 (30%). The treated C/S fabric displays a good dyeability using direct dyes due to the qualified wettability. The mild pH process protects C/S fabrics from damage with a low tensile loss (less than 10%) in warp and weft directions. The WI of C/S fabric mainly depends on the concentration of TBCC/H2O2 couple and radiation time. Moreover, the degradation mechanism of cotton pigment in UVA/TBCC/H2O2 bleaching system was implemented including the identification of effective reactive oxygen species (ROS), tracking of the concentration variation of hydroxyl radical (HO•) under UVA irradiation, and the exploration of degrading kinetics and pathway of morin as the model of pigment impurity. Indicated by the degradation rate constant, both TBCC and UVA plays an accelerating role in enhancing the degradation efficiency. In the radical scavenging experiment, HO• and superoxide radical (O2–•) are two major ROS participating in the degradation. And therein, HO• makes larger contribution than O2–•. HO• concentration has a high positive correlation with radiation duration and TBCC dosage based on the fluorescence labelling method. Confirmed by LC-MS/MS analyses, 2,4,6-trihydroxybenzoic acid (MW: 170.12) and 2,4,-dihydroxybenzoic acid (MW: 154.12) are two major segments degraded from the oxidation and ring-opening of C-ring in morin. Finally, considering energy, water and chemical conservations, the condition i.e. 7.4 g/L of H2O2 (30%) and 20 g/L of TBCC under 30 W UVA radiation for 1 h, is recommended, which significantly increases the WI by 46.5%, and saves about 90% energy and 50% water consumption compared with the conventional waterbath bleaching at 95 °C for 45 min when similar WI enhancement was achieved.

Hetero-Diels-Alder Cycloaddition Reaction of Vinyl Ethers Enables Selective Fluorescent Labeling of Plasmalogens in Human Plasma Lipids.

Plasmalogens (Pls) are vinyl ether-containing glycerophospholipids of broad biological interest. Their abnormal levels are associated with neurological disorders and cardiovascular diseases. The intricacy of analyzing Pls in lipid samples arises from the wide variety of other coexisting lipid species, which underscores the urgent need for a Pls-specific labeling reaction. To address this challenge, we report an efficient hetero-Diels-Alder cycloaddition reaction between nonterminal vinyl ethers of Pls and o-quinolinone quinone methide probes under mild conditions. On the basis of this mechanism, a selective fluorescent labeling method for Pls is developed. The application of this method permits the exclusive derivatization of Pls over other human plasma lipids. The process also imparts labeled Pls with distinct fluorescence emission and chromatographic retention properties. By integrating this method with high-performance liquid chromatography, we are able to identify individual chromatographic signatures of Pls from 10 different human plasma samples. This Pls signature analytical technique, empowered by the Pls-specific labeling reaction, is cost-effective and simple in terms of instrumentation, suggesting its promising potential for the early screening and diagnosis of diseases linked to Pls abnormalities.

Predation of Cyclopoid Copepods on the Theronts of Ichthyophthirius multifiliis: Shedding Light on Biocontrol of White Spot Disease.

White spot disease, caused by the parasitic ciliate Ichthyophthirius multifiliis, is a significant threat to the freshwater fish farming industry worldwide, resulting in massive mortality and economic losses. Eliminating the free-swimming theronts from the culture environment is considered crucial for the control of I. multifiliis infection. It is well-documented that planktonic ciliates are valuable food resources for macro-zooplankton in aquatic ecosystems. In this study, we developed a fluorescence labeling method for alive theronts and found that cyclopoid copepods Thermocyclops taihokuensis, Mesocyclops spp., Macrocyclops sp., and Paracyclopina sp. present predation on the theronts in co-culture experiments. Laboratory challenge tests further confirmed that the presence of zooplankton in the culture water body significantly reduced the infection of I. multifiliis in goldfish (p < 0.01). Results from this study revealed that cyclopoid copepods have the potential to be used as biological control agents against white spot disease in aquaculture.

First person – Nevena Stajković

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Nevena Stajković is first author on ‘ Direct fluorescent labeling of NF186 and NaV1.6 in living primary neurons using bioorthogonal click chemistry’, published in JCS. Nevena is a PhD student in the lab of Ivana Nikić-Spiegel at Centre for Integrative Neuroscience (CIN), Tübingen, Germany, investigating neuronal biology, axon initial segment components (particularly voltage-gated sodium channels) and their role in neurological diseases, fluorescent protein labeling and advanced microscopy methods, such as super-resolution microscopy.

Intracellular RNA and DNA tracking by uridine-rich internal loop tagging with fluorogenic bPNA

The most widely used method for intracellular RNA fluorescence labeling is MS2 labeling, which generally relies on the use of multiple protein labels targeted to multiple RNA (MS2) hairpin structures installed on the RNA of interest (ROI). While effective and conveniently applied in cell biology labs, the protein labels add significant mass to the bound RNA, which potentially impacts steric accessibility and native RNA biology. We have previously demonstrated that internal, genetically encoded, uridine-rich internal loops (URILs) comprised of four contiguous UU pairs (8 nt) in RNA may be targeted with minimal structural perturbation by triplex hybridization with 1 kD bifacial peptide nucleic acids (bPNAs). A URIL-targeting strategy for RNA and DNA tracking would avoid the use of cumbersome protein fusion labels and minimize structural alterations to the RNA of interest. Here we show that URIL-targeting fluorogenic bPNA probes in cell media can penetrate cell membranes and effectively label RNAs and RNPs in fixed and live cells. This method, which we call fluorogenic U-rich internal loop (FLURIL) tagging, was internally validated through the use of RNAs bearing both URIL and MS2 labeling sites. Notably, a direct comparison of CRISPR-dCas labeled genomic loci in live U2OS cells revealed that FLURIL-tagged gRNA yielded loci with signal to background up to 7X greater than loci targeted by guide RNA modified with an array of eight MS2 hairpins. Together, these data show that FLURIL tagging provides a versatile scope of intracellular RNA and DNA tracking while maintaining a light molecular footprint and compatibility with existing methods.

Comparison of CAR-T cell-mediated cytotoxicity assays with suspension tumor cells using high-throughput plate-based image cytometry method

Abstract In the recent decade, chimeric antigen receptor (CAR)-T cell therapy has revolutionized strategies for cancer treatments due to its highly effective clinical efficacy and response for B cell malignancies. Currently, there are six CAR-T cell products available in the market including Kymriah, Yescarta, Tecartus, Breyanzi, Abecma, and Carvykt. The success of CAR-T cell therapy has stimulated the increase in the research and development of various CAR constructs to target different tumor types. Therefore, a robust and efficient in vitro potency assay is needed to quickly identify potential CAR gene design from a library of construct candidates. Traditionally, in vitro CAR-T cell-mediated cytotoxicity is assessed using release assays such as 51Cr (radioactivity), calcein (fluorescence), and LDH (enzymatic). Image cytometry methodologies have been utilized for various CAR-T cell-mediated cytotoxicity assay using different fluorescent labeling methods, mainly due to their ease-of-use, ability to capture cell images for verification, and higher throughput performance. In this work, we employed the Celigo high-throughput plate-based Image Cytometer to evaluate and compare two CAR-T cell-mediated cytotoxicity assays using GFP-expressing or fluorescent dye-labeled myeloma and plasmacytoma cells. Performing time- and E:T ratio-dependent CAR-T cell-mediated cytotoxicity assays, the GFP-based method demonstrated higher sensitivity in detecting the level of cytotoxicity when compared to the CMFDA/DAPI viability method. We have established the criteria and considerations for the selection of cytotoxicity assays that are fit-for-purpose to ensure the results produced are meaningful for the specific testing conditions. None