Strong increase in the autofluorescence of cells signals struggle for survival

Surre, Jérémy; Saint‐Ruf, Claude; Collin, Valérie; Orenga, Sylvain; Ramjeet, Mahendrasingh; Matić, Ivan

doi:10.1038/s41598-018-30623-2

Cited by 114 publications

(107 citation statements)

References 44 publications

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“…Both the protein yield and cell culture density were decreased in the strain containing ThrRS C182A, which is consistent with the previous study (Ling and Söll, 2010). To address potential effects of E. coli cell autofluorescence on GFP fluorescence readings, which could be caused by different cell densities and stress conditions (Mihalcescu et al, 2015;Surre et al, 2018), we used the corresponding stains harboring the same vector but without the reporter gene as controls. Fluorescence readings were subtracted with corresponding backgrounds.…”

Section: Testing the Reporter For Mistranslation Of Threonine Codonssupporting

confidence: 85%

A Synthetic Reporter for Probing Mistranslation in Living Cells

Chen

Ercanbrack

Wang

et al. 2020

Front. Bioeng. Biotechnol.

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Aminoacyl-tRNA synthetases (AARSs) play key roles in maintaining high fidelity of protein synthesis. They charge cognate tRNAs with corresponding amino acids and hydrolyze mischarged tRNAs by editing mechanisms. Impairment of AARS editing activities can reduce the accuracy of tRNA aminoacylation to produce mischarged tRNAs, which cause mistranslation and cell damages. To evaluate the mistranslation rate of threonine codons in living cells, in this study, we designed a quantitative reporter derived from the green fluorescent protein (GFP). The original GFP has multiple threonine codons which could affect the accuracy of measurement, so we generated a GFP variant containing only one threonine residue to specifically quantify mistranslation at the threonine codon. To validate, we applied this single-threonine GFP reporter to evaluate mistranslation at the threonine codon with mutations or modifications of threonine-tRNA synthetase and compared it with other methods of mistranslation evaluation, which showed that this reporter is reliable and facile to use.

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Section: Testing the Reporter For Mistranslation Of Threonine Codonssupporting

confidence: 85%

A Synthetic Reporter for Probing Mistranslation in Living Cells

Chen

Ercanbrack

Wang

et al. 2020

Front. Bioeng. Biotechnol.

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“…Our control results suggest that at 0, 24 and 48 h, when GQD excitation wavelength light is applied, a relatively high level of background fluorescence is present. This background may be from endogenous autofluorescent molecules located in cancer cell mitochondria and lysosomes, which can actively fluoresce when exposed to radiation (27)(28)(29). However, the MB controls did not exhibit higher fluorescence than the PBS blanks suggesting that autofluorescence is not a factor during MB fluorescence analysis.…”

Section: Cell Uptake Measurementmentioning

confidence: 99%

Anticancer Photodynamic Therapy Properties of Sulfur‐Doped Graphene Quantum Dot and Methylene Blue Preparations in MCF‐7 Breast Cancer Cell Culture

Monroe

Belekov

et al. 2019

Photochem & Photobiology

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Photodynamic therapy (PDT) is a field with many applications including chemotherapy. Graphene quantum dots (GQDs) exhibit a variety of unique properties and can be used in PDT to generate singlet oxygen that destroys pathogenic bacteria and cancer cells. The PDT agent, methylene blue (MB), like GQDs, has been successfully exploited to destroy bacteria and cancer cells by increasing reactive oxygen species generation. Recently, combinations of GQDs and MB have been shown to destroy pathogenic bacteria via increased singlet oxygen generation. Here, we performed a spectrophotometric assay to detect and measure the uptake of GQDs, MB and several GQD‐MB combinations in MCF‐7 breast cancer cells. Then, we used a cell counting method to evaluate the cytotoxicity of GQDs, MB and a 1:1 GQD:MB preparation. Singlet oxygen generation in cells was then detected and measured using singlet oxygen sensor green. The dye, H2DCFDA, was used to measure reactive oxygen species production. We found that GQD and MB uptake into MCF‐7 cells occurred, but that MB, followed by 1:1 GQD:MB, caused superior cytotoxicity and singlet oxygen and reactive oxygen species generation. Our results suggest that methylene blue's effect against MCF‐7 cells is not potentiated by GQDs, either in light or dark conditions.

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“…A third autofluorescent component was seen in this cell line, which we consistently observed in ~6% of SUP-B15 cells throughout normal culture, even in unstained cells (Figure S11). Previously, we differentiated such cells, which are likely apoptotic, 2 by localization of the fluorescence signal; strong autofluorescence was observed throughout the cell cytoplasm rather than being confined to the nucleus or membrane. 20 Rather than morphological examination by microscopy or IFC, 5 TDI-SFC can use high spectral resolution to differentiate FITC and 7-AAD emission from this autofluorescent signal.…”

Section: ■Results and Discussionmentioning

confidence: 99%

Time-Delayed Integration–Spectral Flow Cytometer (TDI-SFC) for Low-Abundance-Cell Immunophenotyping

Jackson

2019

Anal. Chem.

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We describe a unique flow cytometer (TDI-SFC) for the immunophenotyping of low-abundance cells, particularly when cell counts are sample-limited and operationally difficult for analysis by fluorescence microscopy (>100 cells) or multiparameter flow cytometry (MFC, <10 000 cells). TDI-SFC combines the high spectral resolution of spectral flow cytometry (SFC) with a CCD operated in time-delayed integration (TDI) for improved duty cycle and sensitivity. Cells were focused with a 1D-sheathing microfluidic device, and fluorescence emission generated from a 488 nm laser was collected by epi-illumination and dispersed along one axis of a CCD by a spectrograph. Along the other axis, the CCD’s shift rate was clocked at a rate that closely matched the cells’ velocity through the field of view. This TDI-SFC format allowed the CCD shutter to remain open during signal acquisition, providing a duty cycle ~100% and assurance that ~95% cells were interrogated. We used fluorescent beads to optimize synchronization of TDI clocking with the sheathed-cell velocity and to improve sensitivity via the excitation intensity, epi-illumination numerical aperture, and integration time. TDI achieved integrated signals of 106 counts at a signal-to-noise ratio (SNR) of 610 for beads corresponding to a load of 4 × 105 antibodies. We also evaluated multiplexing capabilities by spectral deconvolution and undertook a proof-of-concept application to immunophenotype low-abundance cells; the demonstration consisted of immunophenotyping a model cell line, in this case SUP-B15 cells representing B-cell acute lymphoblastic leukemia (B-ALL). The B-ALL cell line was stained against a leukemic marker (terminal deoxynucleotidyl transferase, TdT), and we successfully used spectral unmixing to discriminate TdT(+) cells from TdT(−) cells even at low cell counts (~100 cells). The TDI-SFC could potentially be used in any application requiring the immunophenotyping of low-abundance cells, such as in monitoring measurable residual disease in acute leukemias following affinity enrichment of circulating leukemia cells from peripheral blood.

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Strong increase in the autofluorescence of cells signals struggle for survival

Cited by 114 publications

References 44 publications

A Synthetic Reporter for Probing Mistranslation in Living Cells

A Synthetic Reporter for Probing Mistranslation in Living Cells

Anticancer Photodynamic Therapy Properties of Sulfur‐Doped Graphene Quantum Dot and Methylene Blue Preparations in MCF‐7 Breast Cancer Cell Culture

Time-Delayed Integration–Spectral Flow Cytometer (TDI-SFC) for Low-Abundance-Cell Immunophenotyping

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