Use of a fluorescent membrane probe to identify zooxanthellae in hospite among dissociated endoderm cell culture from coral.
Preparation of homogeneous endoderm cells and culture is a prerequisite to understanding the cellular and molecular mechanism of endosymbiosis in the cnidarian-dinoflagellate association. During the cell isolation from the stony coral Euphyllia glabrescens, various amounts of symbiotic endoderm cells were found to release their symbionts (Symbiodinium spp., or zooxanthellae in generic usage) into the culture. Due to the bulky occupation by zooxanthellae inside the endoderm cell, the symbiotic endoderm cells, or zooxanthellae in hospite, are difficult to be distinguished from released zooxanthellae by microscopic examination. We now report a method for this identification using a fluorescent analogue of sphingomyelin, N-[5-(5,7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-D-erythro-sphingosylphosphorylcholine (C(5)-DMB-SM). Incubation of symbiotic endoderm cells with C(5)-DMB-SM-defatted bovine serum albumin (DF-BSA) complex results in bright fluorescent membrane staining. Nevertheless, the membrane staining of free-living or released zooxanthellae by this complex is significantly decreased or even diminished. This method has provided a fast and reliable assay to identify symbiotic endoderm cells and will greatly accelerate the progress of endosymbiosis research.
Chen
C-S
CS
Symbiosis Research Group, National Museum of Marine Biology and Aquarium, 2 Houwan Road, Checheng, Pingtung, 944 Taiwan, ROC. cchen@nmmba.gov.tw
Lin
H-P
HP
Yeh
C-C
CC
Fang
L-S
LS
D000818:Animals; D037421:Anthozoa / Q000378:metabolism / Q000469:parasitology*; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D002473:Cell Wall / Q000378:metabolism; D002478:Cells, Cultured; D003911:Dextrans / Q000737:chemistry; D004141:Dinoflagellida / Q000302:isolation & purification* / Q000378:metabolism; D004707:Endoderm / Q000378:metabolism / Q000469:parasitology; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013109:Sphingomyelins / Q000737:chemistry; D013559:Symbiosis
D003911:Dextrans; D005456:Fluorescent Dyes; C105705:N-(5-(5,7-dimethyl Bodipy)-1-pentanoyl)-erythro-sphingosylphosphorylcholine; D013109:Sphingomyelins; C015219:fluorescein isothiocyanate dextran; D012710:Serum Albumin, Bovine; D016650:Fluorescein-5-isothiocyanate