An Overview of Flow Cytometry: Its Principles and Applications in Allergic Disease Research

Schmit, Taylor; Klomp, Mitchell; Khan, M. Nadeem

doi:10.1007/978-1-0716-1001-5_13

Cited by 19 publications

(14 citation statements)

References 26 publications

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“…Annexin V binds to the phosphatidylserine (PS) phospholipids, which are translocated to the outer surface of cells during apoptosis [ 138 ]. 7-AAD, on the other hand, is a fluorescent dye that intercalates in double-stranded DNA, with a high affinity for guanine–cytosine residues, and is used as a fluorescent DNA marker in flow cytometry and fluorescence microscopy [ 139 , 140 ]. The PerCP and Pacific Blue channels were used for Annexin V and 7-AAD, respectively, as the emission spectra of these dyes do not overlap; therefore, no compensation is necessary [ 68 ].…”

Section: Methodsmentioning

confidence: 99%

Synergistic Interactions of Cannabidiol with Chemotherapeutic Drugs in MCF7 Cells: Mode of Interaction and Proteomics Analysis of Mechanisms

Alsherbiny

Bhuyan

Low

et al. 2021

IJMS

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Cannabidiol (CBD), a nonpsychoactive phytocannabinoid, has recently emerged as a potential cytotoxic agent in addition to its ameliorative activity in chemotherapy-associated side effects. In this work, the potential interactions of CBD with docetaxel (DOC), doxorubicin (DOX), paclitaxel (PTX), vinorelbine (VIN), and 7-ethyl-10-hydroxycamptothecin (SN−38) were explored in MCF7 breast adenocarcinoma cells using different synergy quantification models. The apoptotic profiles of MCF7 cells after the treatments were assessed via flow cytometry. The molecular mechanisms of CBD and the most promising combinations were investigated via label-free quantification proteomics. A strong synergy was observed across all synergy models at different molar ratios of CBD in combination with SN−38 and VIN. Intriguingly, synergy was observed for CBD with all chemotherapeutic drugs at a molar ratio of 636:1 in almost all synergy models. However, discording synergy trends warranted the validation of the selected combinations against different models. Enhanced apoptosis was observed for all synergistic CBD combinations compared to monotherapies or negative controls. A shotgun proteomics study highlighted 121 dysregulated proteins in CBD-treated MCF7 cells compared to the negative controls. We reported the inhibition of topoisomerase II β and α, cullin 1, V-type proton ATPase, and CDK-6 in CBD-treated MCF7 cells for the first time as additional cytotoxic mechanisms of CBD, alongside sabotaged energy production and reduced mitochondrial translation. We observed 91 significantly dysregulated proteins in MCF7 cells treated with the synergistic combination of CBD with SN−38 (CSN−38), compared to the monotherapies. Regulation of telomerase, cell cycle, topoisomerase I, EGFR1, protein metabolism, TP53 regulation of DNA repair, death receptor signalling, and RHO GTPase signalling pathways contributed to the proteome-wide synergistic molecular mechanisms of CSN−38. In conclusion, we identified significant synergistic interactions between CBD and the five important chemotherapeutic drugs and the key molecular pathways of the combination of CBD and CSN−38 in MCF7 cells. Further in vivo and clinical studies are warranted to evaluate the implementation of CBD-based synergistic adjuvant therapies for breast cancer.

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Section: Methodsmentioning

confidence: 99%

Synergistic Interactions of Cannabidiol with Chemotherapeutic Drugs in MCF7 Cells: Mode of Interaction and Proteomics Analysis of Mechanisms

Alsherbiny

Bhuyan

Low

et al. 2021

IJMS

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“…PS is translocated to the outer surface of cells during apoptosis [87]. Conversely, 7-AAD is a fluorescent dye that intercalates in double-stranded DNA with a high affinity for guanine-cytosine residues and is used as a DNA fluorescent marker in flow cytometry and fluorescence microscopy [88,89].…”

Section: Flow Cytometric Analysis Of Apoptosis In the Mcf7 Cells Using Annexin V-cf Blue And 7aadmentioning

confidence: 99%

Metabolomic Identification of Anticancer Metabolites of Australian Propolis and Proteomic Elucidation of Its Synergistic Mechanisms with Doxorubicin in the MCF7 Cells

Alsherbiny

Bhuyan

Radwan

et al. 2021

IJMS

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The combination of natural products with standard chemotherapeutic agents offers a promising strategy to enhance the efficacy or reduce the side effects of standard chemotherapy. Doxorubicin (DOX), a standard drug for breast cancer, has several disadvantages, including severe side effects and the development of drug resistance. Recently, we reported the potential bioactive markers of Australian propolis extract (AP−1) and their broad spectrum of pharmacological activities. In the present study, we explored the synergistic interactions between AP−1 and DOX in the MCF7 breast adenocarcinoma cells using different synergy quantitation models. Biochemometric and metabolomics-driven analysis was performed to identify the potential anticancer metabolites in AP−1. The molecular mechanisms of synergy were studied by analysing the apoptotic profile via flow cytometry, apoptotic proteome array and measuring the oxidative status of the MCF7 cells treated with the most synergistic combination. Furthermore, label-free quantification proteomics analysis was performed to decipher the underlying synergistic mechanisms. Five prenylated stilbenes were identified as the key metabolites in the most active AP−1 fraction. Strong synergy was observed when AP−1 was combined with DOX in the ratio of 100:0.29 (w/w) as validated by different synergy quantitation models implemented. AP−1 significantly enhanced the inhibitory effect of DOX against MCF7 cell proliferation in a dose-dependent manner with significant inhibition of the reactive oxygen species (p < 0.0001) compared to DOX alone. AP−1 enabled the reversal of DOX-mediated necrosis to programmed cell death, which may be advantageous to decline DOX-related side effects. AP−1 also significantly enhanced the apoptotic effect of DOX after 24 h of treatment with significant upregulation of catalase, HTRA2/Omi, FADD together with DR5 and DR4 TRAIL-mediated apoptosis (p < 0.05), contributing to the antiproliferative activity of AP−1. Significant upregulation of pro-apoptotic p27, PON2 and catalase with downregulated anti-apoptotic XIAP, HSP60 and HIF-1α, and increased antioxidant proteins (catalase and PON2) may be associated with the improved apoptosis and oxidative status of the synergistic combination-treated MCF7 cells compared to the mono treatments. Shotgun proteomics identified 21 significantly dysregulated proteins in the synergistic combination-treated cells versus the mono treatments. These proteins were involved in the TP53/ATM-regulated non-homologous end-joining pathway and double-strand breaks repairs, recruiting the overexpressed BRCA1 and suppressed RIF1 encoded proteins. The overexpression of UPF2 was noticed in the synergistic combination treatment, which could assist in overcoming doxorubicin resistance-associated long non-coding RNA and metastasis of the MCF7 cells. In conclusion, we identified the significant synergy and highlighted the key molecular pathways in the interaction between AP−1 and DOX in the MCF7 cells together with the AP−1 anticancer metabolites. Further in vivo and clinical studies are warranted on this synergistic combination.

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“…At a cost of approximately $10 per sample, flow cytometry can provide a detection limit of 100-300 molecules/single cell, throughput is about 40,000 cells/s, with analytical efficiency of 90%. One issue with flow cytometry is that even the best sorting methods still cannot accurately identify the time dependent changes that are necessary to define the complete metabolome of the sample [46][47][48].…”

Section: Single-cell Technologies 41 Flow Cytometrymentioning

confidence: 99%

Single-Cell Multiomics Analysis for Drug Discovery

Nassar

Raddassi

2021

Metabolites

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Given the heterogeneity seen in cell populations within biological systems, analysis of single cells is necessary for studying mechanisms that cannot be identified on a bulk population level. There are significant variations in the biological and physiological function of cell populations due to the functional differences within, as well as between, single species as a result of the specific proteome, transcriptome, and metabolome that are unique to each individual cell. Single-cell analysis proves crucial in providing a comprehensive understanding of the biological and physiological properties underlying human health and disease. Omics technologies can help to examine proteins (proteomics), RNA molecules (transcriptomics), and the chemical processes involving metabolites (metabolomics) in cells, in addition to genomes. In this review, we discuss the value of multiomics in drug discovery and the importance of single-cell multiomics measurements. We will provide examples of the benefits of applying single-cell omics technologies in drug discovery and development. Moreover, we intend to show how multiomics offers the opportunity to understand the detailed events which produce or prevent disease, and ways in which the separate omics disciplines complement each other to build a broader, deeper knowledge base.

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An Overview of Flow Cytometry: Its Principles and Applications in Allergic Disease Research

Cited by 19 publications

References 26 publications

Synergistic Interactions of Cannabidiol with Chemotherapeutic Drugs in MCF7 Cells: Mode of Interaction and Proteomics Analysis of Mechanisms

Synergistic Interactions of Cannabidiol with Chemotherapeutic Drugs in MCF7 Cells: Mode of Interaction and Proteomics Analysis of Mechanisms

Metabolomic Identification of Anticancer Metabolites of Australian Propolis and Proteomic Elucidation of Its Synergistic Mechanisms with Doxorubicin in the MCF7 Cells

Single-Cell Multiomics Analysis for Drug Discovery

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