forming assay as well as to assay of DNA synthesis (BrdU incorporation), giving an OER (oxygen enhancement ratio) of 2.5 at 60% isoeffect level versus 3.1 for ...
The British Journal of Radiology, 78 (2005), 945–947 DOI: 10.1259/bjr/54004230
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2005 The British Institute of Radiology
Short communication
Resazurin assay of radiation response in cultured cells 1
S ANOOPKUMAR-DUKIE, MSc, 1J B CAREY, BSc, 2T CONERE, MSc, PhD, 2E O’SULLIVAN, BSc, 1 F N VAN PELT, PhD and 1A ALLSHIRE, PhD 1
Department of Pharmacology and Therapeutics, University College Cork, Cork and 2Department of Medical Physics, Cork University Hospital, Wilton, Cork, Ireland
Abstract. We describe use of resazurin reduction for measurement of cell response to irradiation as a simple and non-destructive assay that complements the conventional colony forming assay and can readily be applied to both adherent and non-adherent cell cultures. The resazurin method yields data comparable with the colony forming assay as well as to assay of DNA synthesis (BrdU incorporation), giving an OER (oxygen enhancement ratio) of 2.5 at 60% isoeffect level versus 3.1 for the colony forming assay. Intraday and interday precisions for the resazurin assay were 4.1% and 5.2%, respectively.
Introduction
Materials and methods
The colony-forming assay has long been used to follow survival of irradiated cells and has proved a valuable tool for radiobiologists. However, in recent years several complementary methods, such as the BrdU assay [1], have been used to measure cell response to ionizing radiation. These assays, like most colony-counting protocols, are destructive of samples. Here we describe use of resazurin reduction as a sensitive, reproducible and non-destructive assay of cell response to irradiation that can be used alone or in combination with other methods. Resazurin is a redox dye commonly used as an indicator of chemical cytotoxicity in cultured cells [2]. The assay is based on the ability of viable, metabolically active cells to reduce resazurin to resorufin and dihydroresorufin. This conversion is intracellular [3], facilitated by mitochondrial, microsomal and cytosolic oxidoreductases [2]. Resorufin produced as a result of resazurin bioreduction is measured colorimetrically or fluorometrically. Resazurin is non-toxic to cells and stable in culture medium, allowing continuous measurement of cell proliferation in vitro [4] as either a kinetic or endpoint assay. Toxic insult that impairs cell viability and proliferation also affects the capacity of cultures to reduce resazurin, and the rate of dye reduction is directly proportional to the number of viable cells present [3]. Therefore, as a direct measure of the metabolic competence of cell cultures [5], resazurin reduction may provide a convenient index of cell proliferation following irradiation. Here we compare the resazurin assay to the colonyforming and BrdU incorporation assays in HeLa cells irradiated at doses up to 8 Gy. We show that the resazurin assay can be used in combination with the other methods in the same cell cultures. Precision and linearity range for the assay are also reported.
Chemicals and reagents
Received 1 November 2004 and in final form 22 April 2005, accepted 22 April 2005. Address correspondence to Dr Ashley Allshire.
The British Journal of Radiology, October 2005
Resazurin, crystal violet and Minimal Essential Medium with Eagle’s salts (Hepes modification) were from Sigma Chemical Co. (St. Louis, MO). All other reagents were of the highest purity commercially available.
Cell culture and irradiation The HeLa cell line purchased from American Type Culture Collection through LGC Promochem (Middlesex, UK) was routinely cultured in Minimal Essential Medium with Eagle’s salts (Hepes modification), supplemented with 10% fetal bovine serum, gentamycin (20 mg ml21), Lglutamine (2 mM), sodium bicarbonate (1.5 g l21) and sodium pyruvate (1 mM). Cells (56104) from passages 10– 30 were seeded into PyrexH glass Petri dishes (55 mm2) and incubated for 24 h under standard conditions (5% CO2 in air). The subconfluent cultures were then incubated for 1 h in controlled atmosphere chambers (MIC-101; BillupsRothenburg, Del Mar, CA) under normoxic or hypoxic conditions before irradiation. Hypoxia was generated by infusing the chamber with oxygen free nitrogen (BOC, Ireland) at 10 l min21 for 8 min followed by 2 l min21 for a further 30 min before sealing it. At this point oxygen levels in the medium were below 0.2%, as confirmed using a FOXY Fibre Optic Oxygen Sensor (Ocean Optics, Dunedin, FL). Cells were then irradiated in situ within 10 min at 2 Gy, 3 Gy, 5 Gy or 8 Gy (2 Gy min21), using a GE Saturne Linear Accelerator (Buc, France). These dose measurements were confirmed by thermoluminescent dosimetry and total dose uncertainty was estimated at ¡5%.
Resazurin reduction assay Following irradiations, cells were washed twice with PBS, detached using trypsin (0.25%) and pelleted at 400 g. The soft pellet was then resuspended in fresh culture medium and cells counted using the trypan blue exclusion 945
S Anoopkumar-Dukie, J Carey, T Conere et al
method. Cells were seeded at 56103 cells ml21 into 96 well micro-titre plates and cultured. Culture medium was replaced at day 4. At day 7 it was replaced again, this time supplemented with 44 mM resazurin. 8 h later resazurin reduction was measured colorimetrically (570/ 600 nm) using a Tecan Sunrise plate reader and X-read software (Tecan, Gro¨dig, Austria). Finally, in some experiments cells were washed twice with PBS and used for either the BrdU incorporation or colony-forming assays as described below. Outcomes of these assays were not affected by prior exposure of cell cultures to resazurin.
BrdU incorporation assay This was measured by BrdU ELISA (Roche Diagnostics, East Sussex, UK) according to the manufacturer’s instructions. Briefly, cells labelled with 10 mM BrdU for 4 h at room temperature were fixed and made permeable with the FixDenat solution for 1 h, then incubated with monoclonal anti-BrdU peroxidase-conjugated antibody for 90 min. Cells were washed three times with wash buffer, after which bound peroxidase activity was measured using tetramethyl-benzidine as substrate.
Colony-forming assay Following 25 cm2 tissue with medium washed twice
irradiation, cells were subcultured into culture flasks and incubated for 12 days, changes at days 4 and 8. Cells were then with PBS and stained with 0.1% crystal
violet for 15 min before counting under a light microscope. Clusters of least 50 cells were scored as colonies.
Results and discussion The colony-forming assay has been considered the gold standard for testing the sensitivity of cell lines to ionizing radiation in vitro [6]. Although the introduction of automated colony counters has made the assay easier, it is still time and resource intensive. Proliferation of irradiated cells can also be measured in terms of BrdU incorporation into DNA, but this assay resembles colony counting in that samples are not readily available for further analysis. Figure 1a shows that resazurin reduction, previously used to measure viability of various cell types, including lymphocytes [7] and neonatal neuronal cultures [8], can be used to quantify proliferation of irradiated cells. It yielded an estimate of oxygen enhancement ratio (OER) comparable with that obtained with the colony-forming assay. Dye reduction was directly proportional to cell number up to 50% resazurin conversion to resorufin (data not shown). In a separate study we have shown that the resazurin assay can also be used to detect significant biological effects of radiation at doses as low as 7.5 mGy [9]. In our hands, reproducibility of the resazurin assay is at least as good as that of the other assays; intraday and interday precisions were 4.1% and 5.2%, respectively. However, the resazurin assay is faster, simpler and cheaper. Moreover, since it is non-destructive it can be combined with other assays or endpoints, a key consideration where cell material may be limited. In fact, as
Figure 1. (a) Resazurin reduction, (b) colony formation and (c) BrdU incorporation by HeLa cells 7 days (a, c) or 12 days (b) after irradiation at 2–8 Gy under normoxia (%) or hypoxia (&). Data are means¡standard deviation for three independent experiments. OER, oxygen enhancement ratio calculated at 60% of unirradiated control values.
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The British Journal of Radiology, October 2005
Short communication: Resazurin assay of irradiation injury
few as 80 cells are required to give a reproducible result, a sensitivity comparable with radioactive assays, but without the safety hazards [3]. In conclusion, the resazurin assay may provide a powerful adjunct to the colony-forming assay for the radiobiologist. As a measure of cell proliferation following irradiation it is sensitive, reproducible, fast and economical. Furthermore, it can be combined easily with other endpoints or adapted for high throughput screening.
Acknowledgments This work was supported by Aid Cancer Treatment, Cork. We thank Orla Carey, David Groeger, Eamonn Hayes, Niall McAndrew and Peter O’Keeffe for technical assistance, and the Radiotherapy Department at Cork University Hospital for access to the linear accelerator.
References 1. Joussen AM, Huppertz B, Koch HR, et al. Low-dose-rate ionizing irradiation for inhibition of secondary cataract formation. Int J Radiat Oncol Biol Phys 2001;49:817–25.
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2. Gonzalez RJ, Tarloff JB. Evaluation of hepatic subcellular fractions for Alamar blue and MTT reductase activity. Toxicol In Vitro 2001;15:257–9. 3. O’Brien J, Wilson I, Orton T, Pognan F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 2000;267:5421–6. 4. Zhang HX, Du GH, Zhang JT. Assay of mitochondrial functions by resazurin in vitro. Acta Pharmacol Sin 2004;25:385–9. 5. Magnani E, Bettini E. Resazurin detection of energy metabolism changes in serum-starved PC12 cells and of neuroprotective agent effect. Brain Res Protoc 2000;5:266–72. 6. Pomp J, Wike JL, Ouwerkerk IJ, et al. Cell density dependent plating efficiency affects outcome and interpretation of colony forming assays. Radiother Oncol 1996;40:121–5. 7. Ahmed SA, Gogal RM Jr, Walsh JE. A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay. J Immunol Methods 1994;170:211–24. 8. White MJ, DiCaprio MJ, Greenberg DA. Assessment of neuronal viability with Alamar blue in cortical and granule cell cultures. J Neurosci Methods 1996;70:195–200. 9. Anoopkumar-Dukie S, McMahon A, Allshire A, Conere TJ. Further evidence for biological effects resulting from ionizing radiation doses in the diagnostic X-ray range. Br J Radiol 2005;78:335–7.
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