Observation and quantification of the morphological effect of trypan blue rupturing dead or dying cells
Autoři:
Leo Li-Ying Chan aff001; William L. Rice aff001; Jean Qiu aff001
Působiště autorů:
Department of Advanced Technology R&D, Nexcelom Bioscience LLC., Lawrence, Massachusetts, United States of America
aff001
Vyšlo v časopise:
PLoS ONE 15(1)
Kategorie:
Research Article
doi:
https://doi.org/10.1371/journal.pone.0227950
Souhrn
Trypan blue has long been the gold standard for staining dead cell to determine cell viability. The dye is excluded from membrane-intact live cells, but can enter and concentrate in membrane-compromised dead cells, rendering the cells dark blue. Over the years, there has been an understanding that trypan blue is inaccurate for cell viability under 80% without scientific support. We previously showed that trypan blue can alter the morphology of dead cells to a diffuse shape, which can lead to over-estimation of viability. Here, we investigate the origin of the dim and diffuse objects after trypan blue staining. Utilizing image and video acquisition, we show real-time transformation of cells into diffuse objects when stained with trypan blue. The same phenomenon was not observed when staining cells with propidium iodide. We also demonstrate the co-localization of trypan blue and propidium iodide, confirming these diffuse objects as cells that contain nuclei. The videos clearly show immediate cell rupturing after trypan blue contact. The formation of these diffuse objects was monitored and counted over time as cells die outside of the incubator. We hypothesize and demonstrate that rapid water influx may have caused the cells to rupture and disappear. Since some dead cells disappear after trypan blue staining, the total can be under-counted, leading to over-estimation of cell viability. This inaccuracy could affect the outcomes of cellular therapies, which require accurate measurements of immune cells that will be infused back into patients.
Klíčová slova:
Bright field imaging – Cell enumeration techniques – Cell staining – Fluorescence imaging – Iodides – Membrane staining – Propidium iodide staining – Sucrose
Zdroje
1. Nagata S, Hanayama R, Kawane K (2010) Autoimmunity and the Clearance of Dead Cells. Cell 140(5): 619–630. doi: 10.1016/j.cell.2010.02.014 20211132
2. Rock KL, Kono H (2008) The inflammatory response to cell death. Annual Review of Pathology 3: 99–126. doi: 10.1146/annurev.pathmechdis.3.121806.151456 18039143
3. Zhang Y, Chen X, Gueydan C, Han J (2018) Plasma membrane changes during programmed cell deaths. Cell Research 28: 9–21. doi: 10.1038/cr.2017.133 29076500
4. Arcidiacono JA, Bauer SR, Kaplan DS, Allocca CM, Sarkar S, Lin-Gibson S (2018) FDA and NIST collaboration on standards development activities supporting innovation and translation of regenerative medicine products. Cytotherapy 20: 779–784. doi: 10.1016/j.jcyt.2018.03.039 29784433
5. Page SW (2008) Chapter 10—Antiparasitic drugs. In: Maddison J, Page S, Church D, editors. Small Animal Clinical Pharmacology (Second Edition): Saunders Ltd. pp. 198–260.
6. Piccinini F, Tesei A, Arienti C, Bevilacqua A (2017) Cell Counting and Viability Assessment of 2D and 3D Cell Cultures: Expected Reliability of the Trypan Blue assay. Biological Procedures Online 19(8): 1–12.
7. Beaudoin AR, Kahkonen D (1963) The Effect of Trypan Blue on the Serum Proteins of the Fetal Rat. The Anatomical Record 147(3): 387–395.
8. Black L, Berenbaum MC (1964) Factors Affecting the Dye Exclusion Test for Cell Viability. Experimental Cell Research 35: 9–13. doi: 10.1016/0014-4827(64)90066-7 14190667
9. Gao H-W, Zhao J-F (2003) Interaction of Trypan Blue with Protein and Application. Journal of the Chinese Chemical Society 50: 329–334.
10. Kwok AKH, Yeung C-K, Lai TYY, Chan K-P, Pang CP (2004) Effects of trypan blue on cell viability and gene expression in human retinal pigment epithelial cells. British Journal of Ophthalmology 88(12): 1590–1594. doi: 10.1136/bjo.2004.044537 15548818
11. Mascotti K, McCullough J, Burger SR (2000) HPC viability measurement: trypan blue versus acridine orange and propidium iodide. Transfusion 40(6): 693–696. doi: 10.1046/j.1537-2995.2000.40060693.x 10864990
12. Tennant JR (1964) Evaluation of the Trypan Blue Technique for Determination of Cell Viability. Transplantation 2(6): 686–694.
13. Chan LL-Y, Kuksin D, Laverty DJ, Saldi S, Qiu J (2014) Morphological observation and analysis using automated image cytometry for the comparison of trypan blue and fluorescence-based viability detection method. Cytotech. doi: 10.1007/s10616-014-9704-5 24643390
14. Tran S-L, Puhar A, Ngo-Camus M, Ramarao N (2011) Trypan Blue Dye Enters Viable Cells Incubated with the Pore-Forming Toxin HlyII of Bacillus cereus. PLOS One 6(9): e22876. doi: 10.1371/journal.pone.0022876 21909398
15. Chung DM, Kim JH, Kim JK (2015) Evaluation of MTT and Trypan Blue assays for radiation-induced cell viability test in HepG2 cells. International Journal of Radiation Research 13(4): 1–6.
16. Wainwright M (2010) Dyes, trypanosomiasis and DNA: a historical and critical review. Biotechnic and Histochemistry 85(6): 341–354. doi: 10.3109/10520290903297528 21080764
17. Sarma KD, Ray D, Antony A (2000) Improved sensitivity of trypan blue dye exclusion assay with Ni2+ or Co2+ salts. Cytotechnology 32: 93–95. doi: 10.1023/A:1008144527206 19002971
18. Chan LL, Wilkinson AR, Paradis BD, Lai N (2012) Rapid Image-based Cytometry for Comparison of Fluorescent Viability Staining Methods. Journal of Fluorescence 22(5): 1301–1311. doi: 10.1007/s10895-012-1072-y 22718197
19. Yip A, Webster RM (2018) The market for chimeric antigen receptor T cell therapies. Nature Reviews Drug Discovery 17: 161–162. doi: 10.1038/nrd.2017.266 29375140
20. Department-of-Health-and-Human-Services U.S., Food-and-Drug-Administration, Center-for-Biologics-Evaluation-and-Research (2008) Guidance for FDA Reviewers and Sponsors—Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Somatic Cell Therapy Investigational New Drug Applications (INDs)
21. Hasegawa K, Hosen N (2019) Chimeric antigen receptor T cell therapy for multiple myeloma. Inflammation and Regeneration 39(10): 1–5.
22. Chan LLY, Laverty DJ, Smith T, Nejad P, Hei H, Gandhi R et al. (2013) Accurate measurement of peripheral blood mononuclear cell concentration using image cytometry to eliminate RBC-induced counting error. Journal of Immunological Methods 388(1–2): 25–32. doi: 10.1016/j.jim.2012.11.010 23201386
23. Kofanova OA, Davis K, Glazer B, Souza YD, Kessler J, Betsou F (2014) Viable Mononuclear Cell Stability Study for Implementation in a Proficiency Testing Program: Impact of Shipment Conditions. Biopreservation and Biobanking 12(3): 206–216. doi: 10.1089/bio.2013.0090 24955735
24. Cannon J, Kim D, Maruyama S, Shiomi J (2012) Influence of Ion Size and Charge on Osmosis. Journal of Physical Chemistry B 116(14): 4206–4211.
25. Bursch W, Hochegger K, Török L, Marian B, Ellinger A, Hermann RS (2000) Autophagic and apoptotic types of programmed cell death exhibit different fates of cytoskeletal filaments. Journal of Cell Science 113: 1189–1198. 10704370
26. Ed Costa, Rodrigues EB, Dib E, Penha FM, Furlani BA, Magalhães O. J et al. (2009) Vital Dyes and Light Sources for Chromovitrectomy: Comparative Assessment of Osmolarity, pH, and Spectrophotometry. Investigate Ophthalmology and Visual Science 50(1): 385–391.
27. Lodish H, Berk A, Zipursky SL (2000) Section 15.8 Osmosis, Water Channels, and the Regulation of Cell Volume. Molecular Cell Biology 4th edition. New York: W. H. Freeman.
28. Bischof JC, Padanilam J, Holmes WH, Ezzell RM, Lee RC, Tompkins RG et al. (1995) Dynamics of Cell Membrane Permeability Changes at Supraphysiological Temperatures. Biophysical Journal 68: 2608–2614. doi: 10.1016/S0006-3495(95)80445-5 7647264
29. Mackey BM, Miles CA, Parsons SE, Seymour DA (1991) Thermal denaturation of whole cells and cell components of Escherichia coli examined by differential scanning calorimetry Journal of General Microbiology 137: 2361–2374. doi: 10.1099/00221287-137-10-2361 1722814
30. Altman SA, Randers L, Rao G (1993) Comparison of Trypan Blue Dye Exclusion and Fluorometric Assays for Mammalian Cell Viability Determinations. Biotechnology Progress 9: 671–674. doi: 10.1021/bp00024a017 7764357
31. Solomon M, Wofford J, Johnson C, Regan D, Creer MH (2010) Factors influencing cord blood viability assessment before cryopreservation. Transfusion 50(4): 820–830. doi: 10.1111/j.1537-2995.2009.02491.x 19919556
32. Chan LL-Y, McCulley KJ, Kessel SL (2017) Assessment of Cell Viability with Single-, Dual-, and Multi-Staining Methods Using Image Cytometry. Methods in Molecular Biology. pp. 27–41.
33. Galluzzi L, Baehrecke EH, Chan FK-M (2018) Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death and Differentiation 25: 486–541. doi: 10.1038/s41418-017-0012-4 29362479
Článek vyšel v časopise
PLOS One
2020 Číslo 1
- Proč se v Česku šíří několik „vymýcených“ infekcí najednou? Ptáme se odborníků
- ADHD jako evoluční výhoda? Výzkum se zaměřil na lovce a sběrače
- Není migréna jako migréna: Co pociťují ženy a co muži?
- Metamizol jako analgetikum první volby: kdy, pro koho, jak a proč?
- Není statin jako statin aneb praktický přehled rozdílů jednotlivých molekul
Nejčtenější v tomto čísle
- Severity of misophonia symptoms is associated with worse cognitive control when exposed to misophonia trigger sounds
- Chemical analysis of snus products from the United States and northern Europe
- Calcium dobesilate reduces VEGF signaling by interfering with heparan sulfate binding site and protects from vascular complications in diabetic mice
- Effect of Lactobacillus acidophilus D2/CSL (CECT 4529) supplementation in drinking water on chicken crop and caeca microbiome
Zvyšte si kvalifikaci online z pohodlí domova
Všechny kurzy