Population and single cell metabolic activity of UV-induced VBNC bacteria determined by CTC-FCM and D2O-labeled Raman spectroscopy

Drug resistance and heterogeneous characteristics of human gastric carcinoma cells (BGC823) under the treatment of paclitaxel (PTX) were investigated using single-cell Raman spectroscopy (RS).

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Development of Single Cell Raman Spectroscopy for Cancer Screening and Therapy Monitoring

10.2172/1124918 ◽

2009 ◽

Author(s):

James W. Chan

Keyword(s):

Raman Spectroscopy ◽

Cancer Screening ◽

Single Cell ◽

Therapy Monitoring

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Coarse Raman and optical diffraction tomographic imaging enable label-free phenotyping of isogenic breast cancer cells of varying metastatic potential

10.1101/2020.09.23.309138 ◽

2020 ◽

Author(s):

Santosh Kumar Paidi ◽

Vaani Shah ◽

Piyush Raj ◽

Kristine Glunde ◽

Rishikesh Pandey ◽

...

Keyword(s):

Breast Cancer ◽

Raman Spectroscopy ◽

Molecular Imaging ◽

Single Cell ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Metastatic Potential ◽

Optical Diffraction ◽

Multivariate Curve Resolution ◽

Label Free

AbstractIdentification of the metastatic potential represents one of the most important tasks for molecular imaging of cancer. While molecular imaging of metastases has witnessed substantial progress as an area of clinical inquiry, determining precisely what differentiates the metastatic phenotype has proven to be more elusive underscoring the need to marry emerging imaging techniques with tumor biology. In this study, we utilize both the morphological and molecular information provided by 3D optical diffraction tomography and Raman spectroscopy, respectively, to propose a label-free route for optical phenotyping of cancer cells at single-cell resolution. By using an isogenic panel of cell lines derived from MDA-MB-231 breast cancer cells that vary in their metastatic potential, we show that 3D refractive index tomograms can capture subtle morphological differences among the parental, circulating tumor cells, and lung metastatic cells. By leveraging the molecular specificity of Raman spectroscopy, we demonstrate that coarse Raman microscopy is capable of rapidly mapping a sufficient number of cells for training a random forest classifier that can accurately predict the metastatic potential of cells at a single-cell level. We also leverage multivariate curve resolution – alternating least squares decomposition of the spectral dataset to demarcate spectra from cytoplasm and nucleus, and test the feasibility of identifying metastatic phenotypes using the spectra only from the cytoplasmic and nuclear regions. Overall, our study provides a rationale for employing coarse Raman mapping to substantially reduce measurement time thereby enabling the acquisition of reasonably large training datasets that hold the key for label-free single-cell analysis and, consequently, for differentiation of indolent from aggressive phenotypes.

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The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis

International Journal of Molecular Sciences ◽

10.3390/ijms222312827 ◽

2021 ◽

Vol 22 (23) ◽

pp. 12827

Author(s):

Mahshid Ghasemi ◽

Tyron Turnbull ◽

Sonia Sebastian ◽

Ivan Kempson

Keyword(s):

Cell Viability ◽

Single Cell ◽

Metabolic Activity ◽

Mtt Assay ◽

Single Cell Analysis ◽

Culture Media ◽

Cancer Cell Line ◽

Prostate Cancer Cell Line ◽

Serum Starvation ◽

Treatment Toxicity

The MTT assay for cellular metabolic activity is almost ubiquitous to studies of cell toxicity; however, it is commonly applied and interpreted erroneously. We investigated the applicability and limitations of the MTT assay in representing treatment toxicity, cell viability, and metabolic activity. We evaluated the effect of potential confounding variables on the MTT assay measurements on a prostate cancer cell line (PC-3) including cell seeding number, MTT concentration, MTT incubation time, serum starvation, cell culture media composition, released intracellular contents (cell lysate and secretome), and extrusion of formazan to the extracellular space. We also assessed the confounding effect of polyethylene glycol (PEG)-coated gold nanoparticles (Au-NPs) as a tested treatment in PC-3 cells on the assay measurements. We additionally evaluated the applicability of microscopic image cytometry as a tool for measuring intracellular MTT reduction at the single-cell level. Our findings show that the assay measurements are a result of a complicated process dependant on many of the above-mentioned factors, and therefore, optimization of the assay and rational interpretation of the data is necessary to prevent misleading conclusions on variables such as cell viability, treatment toxicity, and/or cell metabolism. We conclude, with recommendations on how to apply the assay and a perspective on where the utility of the assay is a powerful tool, but likewise where it has limitations.

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