A method for evaluating the use of fluorescent dyes to track proliferation in cell lines by dye dilution

Abstract

Labeling nonquiescent cells with carboxyfluorescein succinimidyl ester (CFSE)-like dyes gives rise to a population width exceeding the threshold for resolving division peaks by flow cytometry. Width is a function of biological heterogeneity plus extrinsic and intrinsic error sources associated with the measurement process. Optimal cytometer performance minimizes extrinsic error, but reducing intrinsic error to the point of facilitating peak resolution requires careful fluorochrome selection and fluorescent cell sorting. In this study, we labeled the Jurkat and A549 cell lines with CFSE, CellTraceViolet (CTV), and eFluor 670 proliferation dye (EPD) to test if we could resolve division peaks in culture after reducing the labeled input widths by cell sorting. Reanalysis of the sorted populations to ascertain the level of reduction achieved always led to widths exceeding the gated limits due to the contribution of errors. Measuring detector-specific extrinsic error by sorting uniform fluorescent particles with similar spectral properties to the tracking dyes allowed us to determine the intrinsic error for each dye and cell type using a simple mathematical approach. We found that cell intrinsic error ultimately dictated whether we could resolve division peaks, and that as this increased, the required sort gate width to resolve any division peaks decreased to the point whereby issues with yield made A549 unsuitable for this approach. Finally, attempts to improve yields by setting two concurrent sort gates on the fluorescence distribution enriched for cells in different stages of the cell cycle that had nonequivalent proliferative properties in culture and thus should be practiced with caution. V C 2013 International Society for Advancement of Cytometry

Frequency and Dynamics of Leukemia-Initiating Cells during Short-term Ex Vivo Culture Informs Outcomes in Acute Myeloid Leukemia Patients

“…S1A-S1C). Moreover, we could not use cell sorting to reduce biologic heterogeneity without the possibility of biasing the cell population studied (10). As AML samples usually have a poor viability at thawing and at later points (see details of influence of cell viability in Supplementary Figs.…”

Appraising the suitability of succinimidyl and lipophilic fluorescent dyes to track proliferation in non-quiescent cells by dye dilution

“…Based on these criteria, we previously found that CellTrace Violet™ (CTV) was the favourable option for working with non-quiescent cells and that e-Flour Proliferation Dye 670™ (EPD) was the least favourable. We also found that certain cell lines were simply at the limit of this approach because the measurement errors associated with analysing such cells by flow cytometry meant that even sorting a narrowed input lead to spreading beyond the limits for peak resolution [20]. In this study, we have extended our appraisal to cover two lipophilic dyes PKH26™ and CellVue Claret™ (CVC) as well as a relatively new red excited version of CTV called CellTrace Far Red™ (CTFR).…”

“…When the dye labelled population of cells is sorted to narrow the input width and re-measured to determine the measurement error as a function of population re-spreading, the dye should make a minimal contribution to any measurement errors. Finally, once in culture it should be well retained and inherited in a symmetrically fashion across the plane of cytokinesis [20]. Based on these criteria, we previously found that CellTrace Violet™ (CTV) was the favourable option for working with non-quiescent cells and that e-Flour Proliferation Dye 670™ (EPD) was the least favourable.…”

“…We have been able to determine detector specific sources of measurement error by sorting uniform fluorescent beads with similar spectral properties to the dyes under investigation and using the width of the re-analysed sorted population as a correction factor [13,20]. As such, we recently developed a method for assessing the suitability of tracking dyes and various cell lines to division tracking by dye dilution following cell sorting to reduce the input widths [20]. The criteria we set out for a good dye was it should label cells brightly, uniformly and with low toxicity.…”

An imaging flow cytometry-based approach to measuring the spatiotemporal calcium mobilisation in activated T cells

“…S7) conforming that our GFP tag did not in any way inhibit the translocation function of NFAT in response to strong signals that bypass the TCR. We then labelled the GFP expressing cells with DiH-Rhod2, MitoDR and DCV to stain the nucleus in live cells with minimal effect on viability (Begum et al, 2013). We then activated these cells with 1 μg/ml CD3 in the presence of Ca 2+ and measured DiH-Rhod2 fluorescence within the MitoDRdefined cell area and correlated this with the degree of NFAT-GFP nuclear translocation for 20 min (Fig.…”