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Adaptation of NS cells growth and differentiation to high-throughput screening-compatible plates.

Garavaglia A, Moiana A, Camnasio S, Bolognini D, Papait R, Rigamonti D, Cattaneo E - BMC Neurosci (2010)

Bottom Line: NS (Neural Stem) cells are a novel population of stem cells that undergo symmetric cell division in monolayer and chemically defined media, while remaining highly neurogenic.This optimized system has also been exploited in homogeneous and high-content assays.Our results show that these mouse NS cells may be suitable for a series of applications in high-throughput format.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dialectica S.r.l., c/o Nerviano Medical Sciences, V.le Pasteur 10, 20014 Nerviano MI, Italy.

ABSTRACT

Background: There is an urgent need of neuronal cell models to be applied to high-throughput screening settings while recapitulating physiological and/or pathological events occurring in the Central Nervous System (CNS). Stem cells offer a great opportunity in this direction since their self renewal capacity allows for large scale expansion. Protocols for directed differentiation also promise to generate populations of biochemically homogenous neuronal progenies. NS (Neural Stem) cells are a novel population of stem cells that undergo symmetric cell division in monolayer and chemically defined media, while remaining highly neurogenic.

Results: We report the full adaptation of the NS cell systems for their growth and neuronal differentiation to 96- and 384-well microplates. This optimized system has also been exploited in homogeneous and high-content assays.

Conclusions: Our results show that these mouse NS cells may be suitable for a series of applications in high-throughput format.

Show MeSH
Homogeneous assays in aNS-1 derived neurons. (A): 3DIV aNS-1 differentiated cells were exposed to H2O2 or STS in 96-well microplates for 24 h. Caspase 3/7 activation, normalized on vitality data as assessed by MTT assay, is reported. Results from one experiment performed in triplicate are shown. Three independent experiments give rise to overlapping results. (B): 21DIV aNS-1 differentiated cells were exposed to Forskolin 250 μM for 15' and cAMP-Glo Assay™ was then performed on 19 well from a 96-well microplate. Row data dispersion around mean value (full line) is reported; upper and lower groups represent untreated control and treated well data respectively; dot lines represent 2SD from mean values: letters in x axes identify each individual well. (C): 21DIV aNS-1 differentiated cells were exposed to adenosine receptor agonist/antagonist for 1 h. Forskolin for 15' was used as positive control. Graph shows cAMP levels modulation, average value from three independent experiments performed in triplicate. * p < 0.05, ** p < 0.01. H2O2: Hydrogen Peroxide; cps, counts per second; STS: staurosporine.
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Figure 4: Homogeneous assays in aNS-1 derived neurons. (A): 3DIV aNS-1 differentiated cells were exposed to H2O2 or STS in 96-well microplates for 24 h. Caspase 3/7 activation, normalized on vitality data as assessed by MTT assay, is reported. Results from one experiment performed in triplicate are shown. Three independent experiments give rise to overlapping results. (B): 21DIV aNS-1 differentiated cells were exposed to Forskolin 250 μM for 15' and cAMP-Glo Assay™ was then performed on 19 well from a 96-well microplate. Row data dispersion around mean value (full line) is reported; upper and lower groups represent untreated control and treated well data respectively; dot lines represent 2SD from mean values: letters in x axes identify each individual well. (C): 21DIV aNS-1 differentiated cells were exposed to adenosine receptor agonist/antagonist for 1 h. Forskolin for 15' was used as positive control. Graph shows cAMP levels modulation, average value from three independent experiments performed in triplicate. * p < 0.05, ** p < 0.01. H2O2: Hydrogen Peroxide; cps, counts per second; STS: staurosporine.

Mentions: Oxidative stress has long been linked to neuronal cell death that is associated with certain neurodegenerative conditions. Neurons are particularly prone to oxidative stress and inadequately equipped with antioxidant defense systems to prevent "ongoing" oxidative damage [25]. As we moved to the optimization of homogenous assays on neuronally differentiated NS cells we have optimized the use of aNS-1 derived neurons as a tool for the detection of hydrogen peroxide induced apoptosis. aNS-1 cells were plated in 96-well microplates and exposed to the differentiation conditions. After three days, cells were exposed to different doses of H2O2 (from 31.25 to 1000 mM) or staurosporine 0.01 mM as positive control and the Caspase-Glo 3/7 Assay (Promega) was performed at the indicated time points. Data were normalized on viability data as assessed by MTT assay, which is more sensitive than ATP assay on differentiated aNS1-cells (data not shown). We observed that, as expected, after 6 h of exposure, staurosporine induces a statistically significant activation of caspases (data not shown). After 24 h, 1 mM and 500 μM H2O2 cause a statistically significant and dose-dependent activation of caspases (Figure 4A). Furthermore, while the control group showed the expected neuronal morphology and dendritic networks, incubation with 250 mM, 500 mM or 1 M H2O2 for 24 h caused a proportional decrease in the number of viable cells (data not shown).


Adaptation of NS cells growth and differentiation to high-throughput screening-compatible plates.

Garavaglia A, Moiana A, Camnasio S, Bolognini D, Papait R, Rigamonti D, Cattaneo E - BMC Neurosci (2010)

Homogeneous assays in aNS-1 derived neurons. (A): 3DIV aNS-1 differentiated cells were exposed to H2O2 or STS in 96-well microplates for 24 h. Caspase 3/7 activation, normalized on vitality data as assessed by MTT assay, is reported. Results from one experiment performed in triplicate are shown. Three independent experiments give rise to overlapping results. (B): 21DIV aNS-1 differentiated cells were exposed to Forskolin 250 μM for 15' and cAMP-Glo Assay™ was then performed on 19 well from a 96-well microplate. Row data dispersion around mean value (full line) is reported; upper and lower groups represent untreated control and treated well data respectively; dot lines represent 2SD from mean values: letters in x axes identify each individual well. (C): 21DIV aNS-1 differentiated cells were exposed to adenosine receptor agonist/antagonist for 1 h. Forskolin for 15' was used as positive control. Graph shows cAMP levels modulation, average value from three independent experiments performed in triplicate. * p < 0.05, ** p < 0.01. H2O2: Hydrogen Peroxide; cps, counts per second; STS: staurosporine.
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Related In: Results  -  Collection

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Figure 4: Homogeneous assays in aNS-1 derived neurons. (A): 3DIV aNS-1 differentiated cells were exposed to H2O2 or STS in 96-well microplates for 24 h. Caspase 3/7 activation, normalized on vitality data as assessed by MTT assay, is reported. Results from one experiment performed in triplicate are shown. Three independent experiments give rise to overlapping results. (B): 21DIV aNS-1 differentiated cells were exposed to Forskolin 250 μM for 15' and cAMP-Glo Assay™ was then performed on 19 well from a 96-well microplate. Row data dispersion around mean value (full line) is reported; upper and lower groups represent untreated control and treated well data respectively; dot lines represent 2SD from mean values: letters in x axes identify each individual well. (C): 21DIV aNS-1 differentiated cells were exposed to adenosine receptor agonist/antagonist for 1 h. Forskolin for 15' was used as positive control. Graph shows cAMP levels modulation, average value from three independent experiments performed in triplicate. * p < 0.05, ** p < 0.01. H2O2: Hydrogen Peroxide; cps, counts per second; STS: staurosporine.
Mentions: Oxidative stress has long been linked to neuronal cell death that is associated with certain neurodegenerative conditions. Neurons are particularly prone to oxidative stress and inadequately equipped with antioxidant defense systems to prevent "ongoing" oxidative damage [25]. As we moved to the optimization of homogenous assays on neuronally differentiated NS cells we have optimized the use of aNS-1 derived neurons as a tool for the detection of hydrogen peroxide induced apoptosis. aNS-1 cells were plated in 96-well microplates and exposed to the differentiation conditions. After three days, cells were exposed to different doses of H2O2 (from 31.25 to 1000 mM) or staurosporine 0.01 mM as positive control and the Caspase-Glo 3/7 Assay (Promega) was performed at the indicated time points. Data were normalized on viability data as assessed by MTT assay, which is more sensitive than ATP assay on differentiated aNS1-cells (data not shown). We observed that, as expected, after 6 h of exposure, staurosporine induces a statistically significant activation of caspases (data not shown). After 24 h, 1 mM and 500 μM H2O2 cause a statistically significant and dose-dependent activation of caspases (Figure 4A). Furthermore, while the control group showed the expected neuronal morphology and dendritic networks, incubation with 250 mM, 500 mM or 1 M H2O2 for 24 h caused a proportional decrease in the number of viable cells (data not shown).

Bottom Line: NS (Neural Stem) cells are a novel population of stem cells that undergo symmetric cell division in monolayer and chemically defined media, while remaining highly neurogenic.This optimized system has also been exploited in homogeneous and high-content assays.Our results show that these mouse NS cells may be suitable for a series of applications in high-throughput format.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dialectica S.r.l., c/o Nerviano Medical Sciences, V.le Pasteur 10, 20014 Nerviano MI, Italy.

ABSTRACT

Background: There is an urgent need of neuronal cell models to be applied to high-throughput screening settings while recapitulating physiological and/or pathological events occurring in the Central Nervous System (CNS). Stem cells offer a great opportunity in this direction since their self renewal capacity allows for large scale expansion. Protocols for directed differentiation also promise to generate populations of biochemically homogenous neuronal progenies. NS (Neural Stem) cells are a novel population of stem cells that undergo symmetric cell division in monolayer and chemically defined media, while remaining highly neurogenic.

Results: We report the full adaptation of the NS cell systems for their growth and neuronal differentiation to 96- and 384-well microplates. This optimized system has also been exploited in homogeneous and high-content assays.

Conclusions: Our results show that these mouse NS cells may be suitable for a series of applications in high-throughput format.

Show MeSH