Management of sedimentation in centrifugal counter-current distribution of sperm cells in an aqueous 2-phase system.
It is generally assumed that centrifugal counter-current distribution (CCCD) in aqueous two-phase systems cannot be employed for analyzing or fractioning cell populations, due to large particles of sediment in the system caused by enhanced gravity. The present work was undertaken to find out whether addition of Percoll to a two-phase system would be a useful method to avoid this cell sedimentation. The results obtained show that bull spermatozoa partition as a unique peak in a CCCD using a Dextran T500-poly(ethylene glycol) 6000 system, and that sedimentation takes place significantly in the upper phase during the process. Addition of increasing concentrations of Percoll made this unique peak wider and two different populations of bull spermatozoa were finally obtained when Percoll concentration rose to 13.6%. This management of cell sedimentation in CCCD could be of great interest for analyzing cell heterogeneity, since the shortening of the time required for counter-current distribution should prevent the loss of cell viability during the separation process. Finally, the results obtained suggest that an increase of viscosity rather than of density is the phase feature which has greater influence on managing cell sedimentation in CCCD.
Pascual
M L
ML
Departamento de Bioquímica, Biología Molecular y Celular, Facultad de Veterinaria, Zaragoza, Spain.
Muiño-Blanco
T
T
Cebrian-Perez
J A
JA
Lopez-Perez
M J
MJ
D000818:Animals; D002417:Cattle; D002498:Centrifugation / Q000379:methods*; D003377:Countercurrent Distribution; D008297:Male; D011205:Povidone; D012822:Silicon Dioxide; D013094:Spermatozoa / Q000166:cytology*; D014867:Water / Q000737:chemistry
D014867:Water; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone