PubMed Health⌕ Search

Biomedical subjects

B F Kimler

Publications and source records attributed to B F Kimler.

111 records · Page 7Linked to original sources
PubMedDate not supplied

Clonogenic assay combined with flow cytometric cell sorting for cell-cycle analysis of human leukemic colony-forming cells.

The purpose of this study is to develop a method combining clonogenic assay and flow cytometric cell sorting based on DNA content for determination of percentage distribution of human malignant clonogenic cells over cell-cycle phases. Flow cytometric cell sorting was carried out for Chinese Hamster Ovary (CHO) and human leukemic bone marrow cells under sterile conditions using a supravital dye for DNA, 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI). The fraction sorted for G0/G1 CHO cells was verified to be almost completely free of G2/M cells and partially cleared of S-phase cells. Cells sorted for G0/G1 fraction were successfully grown as colonies in culture for both CHO and human leukemic marrow cells. The plating efficiency of sorted cells was lower than that of unsorted cells, and the relative contribution of cell damage versus cell selection cannot be determined for this reduction of plating efficiency. The purity of sorted cells must still be improved, and the reason for reduced plating efficiency remains to be defined by further studies. Nonetheless this report represents initial data demonstrating the feasibility of combining two technologies: clonogenic assay and flow cytometric cell sorting based on DNA content. As such, it paves the way for a new approach to studying the cell cycle kinetics of clonogenic cell subpopulations of malignant tissues freshly obtained from patients.

Animals↗
PubMedDate not supplied

Tumor cell-selective flow cytometric analysis for DNA content and cytokeratin expression of clinical tumor specimens by "cross-gating".

A major problem with flow cytometric analysis of clinical solid tumor specimens is that of non-malignant cell contamination which contributes to inaccurate results. Monoclonal antibody to cytokeratin, a marker for epithelial tumor (carcinoma) cells, was successfully used in conjunction with a DNA-specific dye to dually stain specimens so as to obtain DNA profiles exclusively for marker-positive tumor cells. This gating procedure was used also for analysis of the cell marker distribution for those cells having a DNA content consistent with aneuploidy. After successful verification in model systems, clinical carcinoma specimens were studied. The aneuploid population increased from a mean of 61% without gating to a mean of 81% with gating for cytokeratin. The cytokeratin-positive cell fraction increased from a mean of 55% without gating to a mean of 96% with gating for aneuploid DNA content. This gating procedure makes it possible to associate cell markers confidently with tumor cell populations, thus providing objective verification of the tissue of origin for these tumors. It can be performed simultaneously with standard gating for DNA analysis; and both procedures used together, cross-gating, are invaluable for more precise analysis of human tumor DNA and cell markers.

Adenocarcinoma↗