Open AccessAn improved mathematical method to estimate DNA synthesis time of bromodeoxyuridine‐labelled cells, using FCM‐derived data
Author(s) -
Johansson M. C.,
Baldetorp B.,
Bendahl P. O.,
Johansson R.,
Oredsson S. M.
Publication year - 1994
Publication title -
cell proliferation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.647
H-Index - 74
eISSN - 1365-2184
pISSN - 0960-7722
DOI - 10.1111/j.1365-2184.1994.tb01477.x
Subject(s) - propidium iodide , bromodeoxyuridine , labelling , dna , flow cytometry , cell cycle , biology , microbiology and biotechnology , chinese hamster ovary cell , cell , cell culture , biochemistry , genetics , cell growth , apoptosis , programmed cell death
. Chinese hamster ovary cells in vitro were pulse‐labelled with bromodeoxyuridine (BrdUrd and were then allowed to progress through the cell cycle. Every half hour after labelling, cells were harvested and prepared for simultaneous flow cytometric determination of DNA content and incorporated BrdUrd, with the intercalating dye propidium iodide and with a monoclonal antibody against incorporated BrdUrd, respectively. The relative movement (RM), i.e. the relative mean DNA content of the moving cohort of BrdUrd‐labelled cells in relation to that of G 1 and G 2 cells, was calculated. RM was then used to calculate DNA synthesis time (T S ), at all post‐labelling times (t). Since labelled cells in G 2 and mitosis (M) in addition to S phase cells, are included in the cohort of moving labelled cells, and since the time of G 2 and M (T g2+M ) phases is finite, a non‐linear relationship exists between RM and post‐labelling time. Because of this, the use of a linear formula in the calculation of T S yields results that are affected by t. We found that RM data can be corrected with regard to T G2+M resulting in the derivation of a non‐linear T S formula. This non‐linear T S formula gave results that were nearly independent of t. Moreover, windows were set in the mid DNA distributions for G 1 , S and G 2 + M cells in the bivariate DNA v . BrdUrd cytograms, to estimate the fraction of BrdUrd‐labelled cells in each window at every post‐labelling time. Plots of the fraction of BrdUrd‐labelled cells v . post‐labelling time were then made for each window. T S obtained in this way was in agreement with T S obtained with the corrected RM method. In conclusion, we present a method to calculate T s which theoretically first makes the determination of RM independent of T G2+M , and secondly compensates for the non‐linear function of RM with post‐labelling time caused by accumulation of BrdUrd‐labelled cells in G 2 + M.