Quantification of the asymmetric migration of the lipophilic dyes, DiO and DiD, in homotypic co-cultures of chondrosarcoma SW-1353 cells

Lehmann,Tomasz   P.; Juzwa,Wojciech; Filipiak,Krystyna; Sujka‑Kordowska,Patrycja; Zabel,Maciej; Głowacki,Jakub; Głowacki,Maciej; Jagodziński,Paweł   P.

doi:10.3892/mmr.2016.5793

Quantification of the asymmetric migration of the lipophilic dyes, DiO and DiD, in homotypic co-cultures of chondrosarcoma SW-1353 cells

Authors:
- Tomasz P. Lehmann
- Wojciech Juzwa
- Krystyna Filipiak
- Patrycja Sujka‑Kordowska
- Maciej Zabel
- Jakub Głowacki
- Maciej Głowacki
- Paweł P. Jagodziński
View Affiliations

Affiliations: Department of Biochemistry and Molecular Biology, Poznan University of Medical Sciences, 60‑781 Poznan, Poland, Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, 60‑627 Poznan, Poland, Department of Histology and Embryology, Poznan University of Medical Sciences, 60‑781 Poznan, Poland, Medical Center HCP, 61‑485 Poznan, Poland, Department of Paediatric Orthopaedics and Traumatology, Poznan University of Medical Sciences, 61‑545 Poznan, Poland
Published online on: October 4, 2016 https://doi.org/10.3892/mmr.2016.5793
Pages: 4529-4536
Copyright: © Lehmann et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

DiO and DiD are lipophilic cell labelling dyes used in the staining of cells in vivo and in vitro. The aim of the present study was to quantify the asymmetrical distribution of dyes in co‑cultured cells and to measure the intercellular transfer of DiO and DiD. DiO and DiD were applied separately to stain two identical populations of SW‑1353 human chondrosarcoma cells that were subsequently co‑cultured (homotypic co‑culture). The intercellular migration of dyes in the co‑cultured cells was measured by flow cytometry and recorded under a fluorescent microscope. DiD and DiO caused no effect on the proliferation of cells, the degradation rate of the two dyes was comparable and crossover effects between dyes were negligible. The results of the present study suggested that asymmetrical intercellular migration of DiD and DiO was responsible for the asymmetrical distribution of these dyes in co‑cultured cells. To take advantage of the lipophilic dyes migration in the double-stained co-cultured cells we suggest to apply mixed-dyes controls prior to the flow cytometric analysis. These controls are performed by staining cells with a 1:1 mix of the two dyes and would enable the estimation of the intensity of intercellular contact in co‑culture systems. A 1:1 premix of DiO and DiD was applied to estimate cellular effect on intercellular exchange of lipid dyes in co‑cultures incubated with cycloheximide and cytochalasin B. The cellular effect contributed 6‑7% of intercellular migration of the lipophilic dyes, DiO and DiD. The majority of the observed intercellular transfer of these dyes was due to non‑cellular, passive transfer.

View Figures

View References

1	Honig MG and Hume RI: Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures. J Cell Biol. 103:171–187. 1986. View Article : Google Scholar : PubMed/NCBI
2	Progatzky F, Dallman MJ and Lo Celso C: From seeing to believing: Labelling strategies for in vivo cell-tracking experiments. Interface Focus. 3:201300012013. View Article : Google Scholar : PubMed/NCBI
3	Sezgin E, Chwastek G, Aydogan G, Levental I, Simons K and Schwille P: Photoconversion of bodipy-labeled lipid analogues. Chembiochem. 14:695–698. 2013. View Article : Google Scholar : PubMed/NCBI
4	Shapiro HM: Practical Flow Cytometry. 7362003.
5	Rietdorf J and Stelzer EHK: Special optical elementsHandbook of Biological Confocal Microscopy. New York: Springer Verlag; 2006, View Article : Google Scholar
6	Thayanithy V, Dickson EL, Steer C, Subramanian S and Lou E: Tumor-stromal cross talk: Direct cell-to-cell transfer of oncogenic microRNAs via tunneling nanotubes. Transl Res. 164:359–365. 2014. View Article : Google Scholar : PubMed/NCBI
7	Fritzsch B, Muirhead KA, Feng F, Gray BD and Ohlsson-Wilhelm BM: Diffusion and imaging properties of three new lipophilic tracers, NeuroVue Maroon, NeuroVue Red and NeuroVue Green and their use for double and triple labeling of neuronal profile. Brain Res Bull. 66:249–258. 2005. View Article : Google Scholar : PubMed/NCBI
8	Huerta L, López-Balderas N, Larralde C and Lamoyi E: Discriminating in vitro cell fusion from cell aggregation by flow cytometry combined with fluorescence resonance energy transfer. J Virol Methods. 138:17–23. 2006. View Article : Google Scholar : PubMed/NCBI
9	Lo Celso C, Fleming HE, Wu JW, Zhao CX, Miake-Lye S, Fujisaki J, Côté D, Rowe DW, Lin CP and Scadden DT: Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche. Nature. 457:92–96. 2009. View Article : Google Scholar : PubMed/NCBI
10	Maklad A and Fritzsch B: The developmental segregation of posterior crista and saccular vestibular fibers in mice: A carbocyanine tracer study using confocal microscopy. Brain Res Dev Brain Res. 135:1–17. 2002. View Article : Google Scholar : PubMed/NCBI
11	Plotnikov EY, Khryapenkova TG, Galkina SI, Sukhikh GT and Zorov DB: Cytoplasm and organelle transfer between mesenchymal multipotent stromal cells and renal tubular cells in co-culture. Exp Cell Res. 316:2447–2455. 2010. View Article : Google Scholar : PubMed/NCBI
12	Jensen EC: Use of fluorescent probes: Their effect on cell biology and limitations. Anat Rec (Hoboken). 295:2031–2036. 2012. View Article : Google Scholar : PubMed/NCBI
13	Lehmann TP, Filipiak K, Juzwa W, Sujka-Kordowska P, Jagodziński PP, Zabel M, Głowacki J, Misterska E, Walczak M and Głowacki M: Co-culture of human nucleus pulposus cells with multipotent mesenchymal stromal cells from human bone marrow reveals formation of tunnelling nanotubes. Mol Med Rep. 9:574–582. 2014.PubMed/NCBI
14	Burguera EF, Bitar M and Bruinink A: Novel in vitro co-culture methodology to investigate heterotypic cell-cell interactions. Eur Cell Mater. 19:166–179. 2010.PubMed/NCBI
15	Lassailly F, Griessinger E and Bonnet D: ‘Microenvironmental contaminations’ induced by fluorescent lipophilic dyes used for noninvasive in vitro and in vivo cell tracking. Blood. 115:5347–5354. 2010. View Article : Google Scholar : PubMed/NCBI
16	Strassburg S, Hodson NW, Hill PI, Richardson SM and Hoyland JA: Bi-directional exchange of membrane components occurs during co-culture of mesenchymal stem cells and nucleus pulposus cells. PLoS One. 7:e337392012. View Article : Google Scholar : PubMed/NCBI
17	Yumoto K, Berry JE, Taichman RS and Shiozawa Y: A novel method for monitoring tumor proliferation in vivo using fluorescent dye DiD. Cytometry A. 85:548–555. 2014. View Article : Google Scholar : PubMed/NCBI
18	Cirulis JT, Strasser BC, Scott JA and Ross GM: Optimization of staining conditions for microalgae with three lipophilic dyes to reduce precipitation and fluorescence variability. Cytometry A. 81:618–626. 2012. View Article : Google Scholar : PubMed/NCBI
19	Fonseca PC, Nihei OK, Savino W, Spray DC and Alves LA: Flow cytometry analysis of gap junction-mediated cell-cell communication: Advantages and pitfalls. Cytometry A. 69:487–493. 2006. View Article : Google Scholar : PubMed/NCBI
20	Goers L, Freemont P and Polizzi KM: Co-culture systems and technologies: Taking synthetic biology to the next level. J R Soc Interface. 11(pii): 20140065. 2014.PubMed/NCBI
21	Gebauer M, Saas J, Sohler F, Haag J, Söder S, Pieper M, Bartnik E, Beninga J, Zimmer R and Aigner T: Comparison of the chondrosarcoma cell line SW1353 with primary human adult articular chondrocytes with regard to their gene expression profile and reactivity to IL-1beta. Osteoarthritis Cartilage. 8:697–708. 2005. View Article : Google Scholar
22	Agmon A, Yang LT, Jones EG and O'Dowd DK: Topological precision in the thalamic projection to neonatal mouse barrel cortex. J Neurosci. 15:549–561. 1995.PubMed/NCBI