TY - JOUR
T1 - Isolation of cell cycle fractions by counterflow centrifugal elutriation
AU - Kauffman, Michael G.
AU - Noga, Stephen J.
AU - Kelly, Thomas J.
AU - Donnenberg, Albert D.
N1 - Funding Information:
The authors thank Ms. Alicia Russo and Ms. Pamela Rose for expert assistance with cell culture, and Mr. Jim Flook for the cytofluor-ographic analysis. This work was supported in part by Grants GM42780-02 and CA44887 from the National Institutes of Health. M.G.K. is supported by Medical Scientist Training Program Grant 5T32-GM-07309-16.
PY - 1990/11/15
Y1 - 1990/11/15
N2 - Counterflow centrifugal elutriation (CCE) has been used to fractionate cell populations on the basis of sedimentation properties, with minimal perturbation of metabolic function. Therefore, it is an ideal method for the isolation of cell cycle phase specific populations. We present modifications of the standard Beckman centrifugal elutriation system which permit standardization of the elutriation procedure and eliminate inter-run variability. We provide elutriation parameters for the cell cycle fractionation of a variety of cultured cell lines and suggest ways to improve the quality of the cell separations. In addition, we describe protocols for the fractionation of up to 3.50 × 108 cells in the small (JE-6B) Beckman elutriation system. This represents a four- to eight-fold increase in cell numbers over current cell fractionation procedures. Cell cycle populations containing >95% G1, >80% S, and >70% G2 M were consistently obtained using these protocols. Finally, we analyzed phase-enriched fractions from several cultured cell lines for the cell cycle regulation of the enzyme thymidine kinase. The data confirm previous findings that CCE is an excellent means of obtaining physiologically unperturbed cell cycle phase specific fractions.
AB - Counterflow centrifugal elutriation (CCE) has been used to fractionate cell populations on the basis of sedimentation properties, with minimal perturbation of metabolic function. Therefore, it is an ideal method for the isolation of cell cycle phase specific populations. We present modifications of the standard Beckman centrifugal elutriation system which permit standardization of the elutriation procedure and eliminate inter-run variability. We provide elutriation parameters for the cell cycle fractionation of a variety of cultured cell lines and suggest ways to improve the quality of the cell separations. In addition, we describe protocols for the fractionation of up to 3.50 × 108 cells in the small (JE-6B) Beckman elutriation system. This represents a four- to eight-fold increase in cell numbers over current cell fractionation procedures. Cell cycle populations containing >95% G1, >80% S, and >70% G2 M were consistently obtained using these protocols. Finally, we analyzed phase-enriched fractions from several cultured cell lines for the cell cycle regulation of the enzyme thymidine kinase. The data confirm previous findings that CCE is an excellent means of obtaining physiologically unperturbed cell cycle phase specific fractions.
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U2 - 10.1016/0003-2697(90)90384-L
DO - 10.1016/0003-2697(90)90384-L
M3 - Article
C2 - 2077941
AN - SCOPUS:0025221637
SN - 0003-2697
VL - 191
SP - 41
EP - 46
JO - Analytical biochemistry
JF - Analytical biochemistry
IS - 1
ER -