Extracellular calcium does not contribute to cryopreservation-induced cytotoxicity
In vitro cellular & developmental biology : journal of the Tissue Culture Association(1993)
摘要
Summary The possible role of extracellular calcium ([Ca+2]e) in cryopreservation-induced cytotoxicity was tested using Madin-Darby canine kidney (MDCK) cells and a fluorescent multiple
endpoint assay. MDCK cells maintained in 2 mM [Ca+2]e and treated with the calcium ionophore, ionomycin, increased their intracellular calcium ([Ca+2]i) as revealed by the calcium indicator dye, Fluo3 and the bottom-reading spectrofluorometer, CytoFluor 2300. The addition
of 10 mM [ethylene bis (oxyethylenenitrilo)]-tetraacetic acid (EGTA) to the extracellular medium before treatment with ionomycin blocked
this ionomycin-dependent increase in [Ca+2]i. A number of site and activity-specific fluorescent probes were surveyed to determine which indicator dye might best reveal
the ionomycin-induced cytotoxic events during this increase in [Ca+2]i. Although most dyes changed their emission profiles in response to calcium, neutral red was found to best reflect the loss
of [Ca+2]i homeostasis. The NR50 for a 15-min exposure to ionomycin in the presence of 2 mM [Ca+2]e was approximately 2μM ionomycin, but ionomycin had little apparent effect on neutral red retention when 10 mM EGTA was added to the extracellular medium. Thus it was clear that an increase in [Ca+2]i could be cytotoxic to MDCK cells and that neutral red could monitor this cytotoxic episode. To test if [Ca+2]e was similarly cytotoxic during cryopreservation, MDCK cells were subjected to cryopreservation in the presence of dimethylsulfoxide
(DMSO). In contrast to previous studies, plasma membrane integrity, not lysosomal function, seemed to best correlate with
cell survival subsequent to cryopreservation. In addition, decreasing [Ca+2]e had no discernable effect on the retention of plasma membrane indicator dyes, neutral red, or cell survival. It is concluded
that a) plasma membrane indicator dyes, not neutral red, might be better indicators of cytotoxicity occurring during cryopreservation;
b) DMSO might be toxic to lysosomes during cryopreservation of cultured cells; and c) although [Ca+2]e can contribute to cytotoxicity, the presence of [Ca+2]e might not influence cryopreservation-induced cytotoxicity.
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关键词
fluo3,multiple endpoint.,cryopreservation,calcein,cfda,calcium cytotoxicity,cytofluor 2300,neutral red,intracellular calcium
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