Measurement of Cell Death in Mammalian Cells

Cummings, Brian S.; Wills, Lauren P.; Schnellmann, Rick G.

doi:10.1002/0471141755.ph1208s25

Cited by 203 publications

(165 citation statements)

References 60 publications

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“…The extracellular LDH release used in this regard is a well-established biomarker of cell membrane integrity commonly used for quantifying cell death. 25,26 Furthermore, lactate and ammonium were substantially reduced in all the low Q and N, higher E and D media (Figures 5C,D). Cell-specific productivities were also improved, and the titer levels were also slightly higher than the control medium despite reduced VCD levels ( Figures 5E,F).…”

Section: Rebalance Of Energy Metabolism Amino Acids To Improve Q P Anmentioning

confidence: 93%

Principles and approach to developing mammalian cell culture media for high cell density perfusion process leveraging established fed‐batch media

Lin

Leighty

Godfrey

et al. 2017

Biotechnology Progress

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Perfusion medium was successfully developed based on our fed-batch platform basal and feed media. A systematic development approach was undertaken by first optimizing the ratios of fed-batch basal and feed media followed by targeted removal of unnecessary and redundant components. With this reduction in components, the medium could then be further concentrated by 2× to increase medium depth. The medium osmolality was also optimized where we found ∼360 mOsm/kg was desirable resulting in a residual culture osmolality of ∼300 mOsm/kg for our cell lines. Further building on this, the amino acids Q, E, N, and D were rebalanced to reduce lactate and ammonium levels, and increase the cell-specific productivity without compromising on cell viability while leaving viable cell density largely unaffected. Further modifications were also made by increasing certain important vitamin and lipid concentrations, while eliminating other unnecessary vitamins. Overall, an effective perfusion medium was developed with all components remaining in the formulation understood to be important and their concentrations increased to improve medium depth. The critical cell-specific perfusion rate using this medium was then established for a cell line of interest to be 0.075 nL/cell-day yielding 1.2 g/L-day at steady state. This perfusion process was then successfully scaled up to a 100 L single-use bioreactor with an ATF6 demonstrating similar performance as a 2 L bioreactor with an ATF2. Large volume handling challenges in our fed-batch facility were overcome by developing a liquid medium version of the powder medium product contained in custom totes for plug-and-play use with the bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:891-901, 2017.

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Section: Rebalance Of Energy Metabolism Amino Acids To Improve Q P Anmentioning

confidence: 93%

Principles and approach to developing mammalian cell culture media for high cell density perfusion process leveraging established fed‐batch media

Lin

Leighty

Godfrey

et al. 2017

Biotechnology Progress

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“…PI enters only into cells with a damaged cell membrane and is considered to be primarily a marker of necrotic cells [99]. OHCs were placed in media containing 2 μM PI [100, 101] for 2 h prior to imaging.…”

Section: Methodsmentioning

confidence: 99%

Acute death of astrocytes in blast-exposed rat organotypic hippocampal slice cultures

et al. 2017

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Blast traumatic brain injury (bTBI) affects civilians, soldiers, and veterans worldwide and presents significant health concerns. The mechanisms of neurodegeneration following bTBI remain elusive and current therapies are largely ineffective. It is important to better characterize blast-evoked cellular changes and underlying mechanisms in order to develop more effective therapies. In the present study, our group utilized rat organotypic hippocampal slice cultures (OHCs) as an in vitro system to model bTBI. OHCs were exposed to either 138 ± 22 kPa (low) or 273 ± 23 kPa (high) overpressures using an open-ended helium-driven shock tube, or were assigned to sham control group. At 2 hours (h) following injury, we have characterized the astrocytic response to a blast overpressure. Immunostaining against the astrocytic marker glial fibrillary acidic protein (GFAP) revealed acute shearing and morphological changes in astrocytes, including clasmatodendrosis. Moreover, overlap of GFAP immunostaining and propidium iodide (PI) indicated astrocytic death. Quantification of the number of dead astrocytes per counting area in the hippocampal cornu Ammonis 1 region (CA1), demonstrated a significant increase in dead astrocytes in the low- and high-blast, compared to sham control OHCs. However only a small number of GFAP-expressing astrocytes were co-labeled with the apoptotic marker Annexin V, suggesting necrosis as the primary type of cell death in the acute phase following blast exposure. Moreover, western blot analyses revealed calpain mediated breakdown of GFAP. The dextran exclusion additionally indicated membrane disruption as a potential mechanism of acute astrocytic death. Furthermore, although blast exposure did not evoke significant changes in glutamate transporter 1 (GLT-1) expression, loss of GLT-1-expressing astrocytes suggests dysregulation of glutamate uptake following injury. Our data illustrate the profound effect of blast overpressure on astrocytes in OHCs at 2 h following injury and suggest increased calpain activity and membrane disruption as potential underlying mechanisms.

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“…Having shown that TBK1 overexpression reduced mutant HTTex1 aggregates in HEK293T cells, we sought to determine whether there would also be suppression of mutant HTTex1 cytotoxicity. We used a previously established cytotoxicity (cell death) assay based on the measurement of nuclear condensation (Cummings and Schnellmann, 2004) during neuronal cell death induced by the expression of mutant HTT (Arbez et al, 2017).…”

Section: Consistent With Our Observation Inmentioning

confidence: 99%

TBK1 regulates autophagic clearance of soluble mutant huntingtin and inhibits aggregation/toxicity in different models of Huntington’s disease

Hegde

Chiki

Petricca³

et al. 2019

Preprint

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Phosphorylation of the N-terminal domain of the Huntingtin (HTT) protein (at T3, S13, and S16) has emerged as a key regulator of HTT stability, clearance, localization, aggregation and toxicity. Herein, we report the discovery and validation of a kinase, TANK-binding kinase 1 (TBK1), that specifically and efficiently phosphorylates both wild-type and mutant full-length or N-terminal fragments of HTT in vitro (S13/S16) and in cell/ neuronal cultures (S13). We show that overexpression of TBK1 in mammalian cells, primary neurons and a Caenorhabditis elegans model of Huntington's Disease (HD) increases mutant HTTex1 phosphorylation, lowers its levels, increases its nuclear localization and significantly reduces its aggregation and cytotoxicity. Our mechanistic studies demonstrate that the TBK1-mediated neuroprotective effects are due to phosphorylation-dependent inhibition of mutant HTTex1 aggregation and an increase in autophagic flux. These findings suggest that upregulation and/or activation of TBK1 represents a viable strategy for the treatment of HD.3

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Measurement of Cell Death in Mammalian Cells

Cited by 203 publications

References 60 publications

Principles and approach to developing mammalian cell culture media for high cell density perfusion process leveraging established fed‐batch media

Principles and approach to developing mammalian cell culture media for high cell density perfusion process leveraging established fed‐batch media

Acute death of astrocytes in blast-exposed rat organotypic hippocampal slice cultures

TBK1 regulates autophagic clearance of soluble mutant huntingtin and inhibits aggregation/toxicity in different models of Huntington’s disease

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