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Conditioned medium from apoptotic cardiomyocytes stimulated EPC migration, while conditioned medium from senescent cardiomyocytes did not produce any effect (Fig. 5).
We also found that conditioned medium from doxorubicin-induced senescent cardiomyocytes does not attract untreated EPCs, unlike conditioned medium from apoptotic cardiomyocytes which has a strong chemoattractant capacity.
To obtain an alternatively activated phenotype, macrophages were stimulated with conditioned medium from apoptotic T cells (CM).
To investigate the probability of Aβ-induced membrane damage, we have assessed the protective effect of the ethyl acetate fraction using the LDH assay, measuring the activity of this stable enzyme released into the medium from apoptotic PC12 cells.
To this end, culture medium from apoptotic HCT116 cells was immuno-precipitated with purified NEAs, and probed with the cleaved CK-18 antibody, M30, or with an antibody recognizing cleaved PARP.
Because the neuronal plasma membrane is sensitive to oxidative stress, the cell membrane protective effect of the NAF derived from black soybean seed coat extracts on Aβ-induced neurotoxicity was investigated by the LDH release assay, measuring the activity of this stable enzyme released into the medium from apoptotic PC 12 cells.
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We therefore investigated the effect of conditioned medium from doxorubicin-induced apoptotic or senescent H9c2 cardiomyocytes on EPC migration.
In order to obtain conditioned medium from senescent or apoptotic H9c2, we treated the cells for 3 hours with 0.1 or 1 µM doxorubicin, respectively, in growth factor-free DMEM plus 0.1% BSA, then the H9c2 were grown in fresh medium (growth factor-free DMEM plus 0.1% BSA) for 24 hours [9].
As a pre-requisite step for validation as plasma biomarkers for apoptosis, it was important to determine whether purified NEAs could bind caspase-cleavage products, such as cleaved CK-18, secreted into the medium from cultures of apoptotic cells.
It is possible that the high percentage of serum (10%) in the culture medium protected both cultures from apoptotic cell death, therefore maintaining the high viabilities recorded.
Chronic incubation of neurons with elevated concentrations of KCl in the culture medium has been shown to protect neurons from apoptotic cell death resulting from over-inhibition and from neurotrophic factor deprivation.
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