Application of the CYTOMIC 12 flow cytometric compact analyzer for automatic kinetic measurements

Abstract

Flow cytometry, usually applied to cells which have time independent features, can also be used for kinetic experiments where the change of cell populations with time is investigated. Dedicated time sequencing programs written in Assembler and incorporated in the CYTOMIC 12 analyzer (4) are described. A sequence of 64 one parameter histograms can be automatically acquired and immediately displayed as a pseudo-twoparameter histogram. The acquisition time for each of the subsequent histograms can be selected between 1 and 32 seconds. Kinet- ~_ _~Flow cytometric techniques have been used to study time dependent changes of biological cells (1, 6, 10, 11). For fast kinetic experiments in the time range of seconds, special transducers are required where the cells are directly introduced from the reaction vessel into the sensing zone of the flow cytometer ( 5 ) . At the same time, the requirements for the data acquisition and processing equipment differ from the requirements for conventional systems in which the cells are considered to have features that are stable with time. The data system must be able to measure, store, and display automatically a large number of data which are coordinated to the different time phases of the cell kinetics under consideration. By composing suitably the data for the different time phases, the course of the cell kinetics can be reconstructed.In general two methods can be applied in flow cytometric time kinetics:First method: With the histogram method, cell features are measured and classified in histograms over distinct time intervals which are normally equal in length. The absolute length of the intervals depends on the rate of change of the cells being investigated. For example, cell change which lasts 60 sec may be observed over 60 time intervals of 1 sec each, and with a pulse rate of 2000 pulses/sec, an average of 2000 cells would be investigated and stored in the form of a histogram during each time interval.ics lasting up to 34 minutes are resolved into 64 time intervals. Two parameter kinetics can be resolved into 12 32 X 32 channel, two parameter histograms which are displayed and evaluated immediately on the analyzer screen in groups of 4 without using complicated list mode procedures. The standard CYTOMIC 12 software can be applied for processing and printing of the sequence distribution curves.

Effect of 5‐fluoro‐2′‐deoxyuridine (FdUrd) on 5‐bromo‐2′‐deoxyuridine (BrdUrd) incorporation into DNA, measured with a monoclonal BrdUrd antibody and by the BrdUrd/hoechst quenching effect

“…The fluorescence intensity of the G1 peak was used for Calibration on both axes. The biparameter distributions of the red DNA and blue dThd fluorescence were stored in a 64 x 64 channel memory of a Cytomic 12 (19).…”

Measurement of efflux from G1-phase in a growth factor dependent cell line

“…Two fluorescence intensities of each cell were measured by using an ICP-22 (Fhywe, G6ttingen, FRG) bivariate flow cytometer linked to a Cytomic-12 pulse height analyzer (Kachel et al, 1983). The 2-dimensional fluorescence distributions were stored by a PDP-11 computer and analyzed interactively on a graphical terminal enhanced with a light pen by using self-developed software.…”

Flow cytometry analysis of recombinant Saccharomyces cerevisiae populations

“…Enzyme kinetic assays were ear correlation between plasmid copy number and 0-performed with the automatic data acquisition mode of galactosidase activity may break down. Establishing the the CYTOMIC 12 enabling automatic data acquisition quantitative nature of this gene number-gene product for 5 s in 10-s intervals (17). activity relationship requires independent data.…”