Cells and Holograms – Holograms and Digital Holographic Microscopy as a Tool to Study the Morphology of Living Cells

We present a method to study the morphology of living, dividing and dying cells using DHM. DHM is a non-invasive, non-destructive and non-phototoxic method which allows the user to perform both qualitative and quantitative measurements of living cells over time. We show here our results on cell division and cell death in single cells. The morphological analyses performed here show changes caused by cell death and cell division, and indicate the possibilities to discriminate between different types of cell death. Cells dying in an apoptosis-like manner display different cell area and cell thickness profiles over time compared to cells dying in a necrosis-like manner, although their volume profiles are very similar. Dividing cells show a characteristic dip in the volume profile, which makes them easily distinguishable. Also, several previous studies show the versatile abilities of DHM. Different cell types have been studied and the morphology has been used to determine cell functionality as well as changes in morphology related to the environment. Cell morphology parameters can be very useful when following the effects of different treatments, the process of differentiation as well as cell growth and cell death. Cell morphology studied by DHM can be useful in toxicology, stem cell and cancer research

The BRAF Inhibitor Vemurafenib Activates Mitochondrial Metabolism and Inhibits Hyperpolarized Pyruvate–Lactate Exchange in BRAF-Mutant Human Melanoma Cells

Understanding the impact of BRAF signaling inhibition in human melanoma on key disease mechanisms is important for developing biomarkers of therapeutic response and combination strategies to improve long-term disease control. This work investigates the downstream metabolic consequences of BRAF inhibition with vemurafenib, the molecular and biochemical processes that underpin them, their significance for antineoplastic activity, and potential as noninvasive imaging response biomarkers. H-1 NMR spectroscopy showed that vemurafenib decreases the glycolytic activity of BRAF-mutant (WM266.4 and SKMEL28) but not BRAF(WT) (CHL-1 and D04) human melanoma cells. In WM266.4 cells, this was associated with increased acetate, glycine, and myo-inositol levels and decreased fatty acyl signals, while the bioenergetic status was maintained. C-13 NMR metabolic flux analysis of treated WM266.4 cells revealed inhibition of de novo lactate synthesis and glucose utilization, associated with increased oxidative and anaplerotic pyruvate carboxylase mitochondrial metabolism and decreased lipid synthesis. This metabolic shift was associated with depletion of hexokinase 2, acyl-CoA dehydrogenase 9, 3-phosphoglycerate dehydrogenase, and monocarboxylate transporters (MCT) 1 and 4 in BRAF-mutant but not BRAF(WT) cells and, interestingly, decreased BRAF-mutant cell dependency on glucose and glutamine for growth. Further, the reduction in MCT1 expression observed led to inhibition of hyperpolarized C-13-pyruvatelactate exchange, a parameter that is translatable to in vivo imaging studies, in live WM266.4 cells. In conclusion, our data provide new insights into the molecular and metabolic consequences of BRAF inhibition in BRAF-driven human melanoma cells that may have potential for combinatorial therapeutic targeting as well as noninvasive imaging of response. (C) 2016 AACR

Evaluation of MRS detectable metabolic markers of MEK1/" targeted therapies

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Digital holographic microscopy for non-invasive monitoring of cell cycle arrest in l929 cells.

Digital holographic microscopy (DHM) has emerged as a powerful non-invasive tool for cell analysis. It has the capacity to analyse multiple parameters simultaneously, such as cell- number, confluence and phase volume. This is done while cells are still adhered and growing in their culture flask. The aim of this study was to investigate whether DHM was able to monitor drug-induced cell cycle arrest in cultured cells and thus provide a non-disruptive alternative to flow cytometry. DHM parameters from G1 and G2/M cell cycle arrested L929 mouse fibroblast cells were collected. Cell cycle arrest was verified with flow cytometry. This study shows that DHM is able to monitor phase volume changes corresponding to either a G1 or G2/M cell cycle arrest. G1-phase arrest with staurosporine correlated with a decrease in the average cell phase volume and G2/M-phase arrest with colcemid and etoposide correlated with an increase in the average cell phase volume. Importantly, DHM analysis of average cell phase volume was of comparable accuracy to flow cytometric measurement of cell cycle phase distribution as recorded following dose-dependent treatment with etoposide. Average cell phase volume changes in response to treatment with cell cycle arresting compounds could therefore be used as a DHM marker for monitoring cell cycle arrest in cultured mammalian cells

MTS cell viability measurement of dose-dependent etoposide induced G2/M arrest.

<p>L929 cells treated with 0.1–10 µM etoposide (ETO) for 24 h. <b>A.</b> MTS analysis show a dose-dependent shift in cell viability post-etoposide treatment as compared to controls. All results are significant except # which indicates P>0.05 compared to controls, n3.</p

Digital holographic microscopy of staurosporine induced G1 arrest and etoposide or colcemid induced G2/M arrest.

<p>L929 cells treated with 20 nM staurosporine (STS) or 3 µM colcemid (COL) or 1 µM etoposide (ETO) for 24 h (untreated controls, C). <b>A.</b> Digital holographic microscopy images, artificially coloured, with increasing thickness shown as greenB. STS treatment has no significant effect on average cell numbers; however it decreases average confluence and average cell volume. COL treatment has no significant effect on average cell number, instead average cell confluence decreases and average cell volume increases. ETO treatment significantly lowers average cell numbers, average confluence and increases average cell volume. Images and histograms are representative of n3. All results are significant except # which indicates P>0.05 compared to controls.</p

Flow cytometric measurement of dose-dependent etoposide induced G2/M arrest.

<p>L929 cells treated with 0.1–10 µM etoposide (ETO) for 24 h (untreated controls, C). <b>A.</b> DNA histograms obtained from flow cytometry measurements show a dose-dependent shift in the G1 to G2/M cell cycle phase. <b>B.</b> ETO increases G2/M phase population of cells and decreases S and G1 population of cells. Images and histograms are representative of n3. All results are significant except # which indicates P>0.05 compared to controls.</p

Digital holographic microscopy of dose-dependent etoposide induced G2/M arrest.

<p>L929 cells treated with 0.1–10 µM etoposide (ETO) for 24 h (untreated controls, C). <b>A.</b> Digital holographic microscopy images of a dose-dependent increase in the average cell volume. Images are artificially coloured, with increasing thickness shown as greenB. ETO reduces average cell number, decreases average cell confluence and increases average cell volume. Images and histograms are representative of n3. All results are significant except # which indicates P>0.05 compared to controls.</p

Flow cytometric measurement of staurosporine induced G1 arrest and colcemid or etoposide induced G2/M arrest.

<p>L929 cells treated with 20 nM staurosporine (STS) or 3 µM colcemid (COL) or 1 µM etoposide (ETO) for 24 h (untreated controls, C). <b>A.</b> DNA histograms obtained from flow cytometry measurements show a shift in the G2/M to G1 cell cycle phase for STS treatment and reversed for COL or ETO, a G1 to G2/M shift. <b>B.</b> STS increases G1 phase population of cells and COL and ETO increases G2/M phase population of cells. Images and histograms are representative of n3. All results are significant, P≤0.05 compared to controls.</p