Chapter 4 the Use of Bromodeoxyuridine Incorporation

Chapter 4 The Use of Bromodeoxyuridine Incorporation Assays to Assess Corneal Stem Cell Proliferation Ashley M. Crane and Sanjoy K. Bhattacharya Abstract Bromodeoxyuridine (BrdU) incorporation assays have long been used to detect DNA synthesis in vivo and in vitro. The key principle of this method is that BrdU incorporated as a thymidine analog into nuclear DNA represents a label that can be tracked using antibody probes. In this chapter, we describe BrdU incorporation into limbal stem cells. The colorimetric reaction produced by this assay can be detected by immunohistochemistry, and using appropriate controls, it can be used for determination of proliferating properties of restricted progenitor cells derived from the cornea. Key words Bromodeoxyuridine , 5-Bromo-2 ¢ -deoxyuridine , Thymidine analog , Bromodeoxyuridine incorporation assay 1 Introduction 5-Bromo-2¢ -deoxyuridine (bromodeoxyuridine (BrdU)) is a thymidine analog which differs from thymidine in its substitution of bromine for a methyl group. BrdU has been in use since the 1980s to label proliferating and migrating cells in various human and animal organs [ 1 ] . Since this time, BrdU incorporation has been reported as a method to detect DNA synthesis in over 20,000 scienti fi c studies [ 2 ] . The use of this halopyrimidine is preferred over the older technique involving the use of 3 H-Thymidine ( 3 H- dT) autoradiography, due to the lower cost, faster processing time, and nonradioactive properties of BrdU [ 3 ] . Another advantage of using BrdU for labeling is that although this chemical can be con- sidered to be toxic [3 ] , it is usually not so at the low concentrations used for labeling [ 4 ] . BrdU competes with thymidine for incorporation into nuclear DNA during the S-phase of the cell cycle [ 1 ] . It is important, how- ever, to note that BrdU can also be incorporated into nuclear DNA during cell repair or cell degeneration [ 5 ] , but the amount of analog Bernice Wright and Che J. Connon (eds.), Corneal Regenerative Medicine: Methods and Protocols, Methods in Molecular Biology, vol. 1014, DOI 10.1007/978-1-62703-432-6_4, © Springer Science+Business Media New York 2013 65 66 Ashley M. Crane and Sanjoy K. Bhattacharya expected to be incorporated into DNA in these situations is much lower than that expected during cell division [ 5 ] . Therefore, BrdU serves as a marker of DNA synthesis, and separate means such as counting mitotic fi gures may be employed to ensure accuracy with regard to cell division [ 5, 6 ] . After incorporation into nuclear DNA, BrdU can be detected with immunohistochemical methods. One limitation of this method is that, unlike 3 H-dT autoradiography, BrdU immunohistochemistry is not stoichiometric. That is, BrdU labeling relies heavily on the accuracy of detection techniques and therefore cannot be used for absolute quanti fi cation [ 5 ] . After BrdU is incorporated into nuclear DNA, samples are fi xed and incubated with anti-BrdU monoclonal antibodies and nucleases (or they are exposed to heat or other conditions which cause DNA denaturation). This denaturation is necessary to allow anti-BrdU monoclonal antibodies to gain access to the incorporated BrdU in single stranded DNA. The sample is sub- sequently incubated with a secondary antibody against the anti- BrdU antibody. The secondary antibody is often associated with an enzyme, which when exposed to its added substrate, causes a colorimetric reaction. The reaction can be observed by bright- fi eld microscopy. BrdU has a history of use in corneal limbal stem cells, and has been applied in vivo in both dividing and slow cycling limbal cells [ 7 ] . The thymidine analog has also been used in double labeling protocols in limbal cells [ 8 ] and therefore can be combined with other experiments designed to detect these cells. BrdU incorporation assays can be used to investigate the proliferation of adherent and suspension cells as well as cell cycling in tissue sections. 2 Materials Store all materials at room temperature or as recommended by the manufacturer. Dispose of materials using established disposal protocols. 1. Ethanol fi xative: add 50 mM glycine solution to 70 mL absolute ethanol for a total of 100 mL of ethanol fi xative, adjust to pH 2.0, and store at 4 °C. 2. Substrate buffer: prepare 100 mM Tris–HCl-buffer, 100 mM NaCl, 50 mM MgCl2 , adjust to pH 9.5 (at 20 °C), and store at 4 °C. 3. Sterile culture medium (media that is typically used to culture limbal stem cells are discussed in Chapters 3 and 6 ). 4. Plastic slide box. 5. Phosphate buffered saline (PBS) (1×). 6. Double distilled water. The Use of Bromodeoxyuridine Incorporation Assays... 67 7. Glycerol gelatin aqueous slide mounting medium (cat. no. GG1, Sigma Aldrich). 8. Fat-free, poly- L -lysine coated glass slides for frozen tissue sections. 9. Cryostat for preparing tissue sections. 10. BrdU Labeling and Detection Kit II (cat. no. 11299964001, Roche Diagnostics) containing: (a) BrdU labeling reagent (1,000× concentrate). For in vitro labeling, prepare BrdU labeling reagent immediately prior to use. Dilute BrdU labeling reagent with sterile cell culture medium at 1:1,000 to create BrdU labeling solution. Do not store. For in vivo labeling, do not dilute BrdU labeling reagent. Prepare 2 mL/100 g body weight of the animal. (b) Washing buffer concentrate (10× concentrate). Dilute washing buffer concentrate with double distilled water at 1:10 to create working washing buffer. Store at 4 °C. (c) Incubation Buffer. (d) Anti-BrdU antibody containing nucleases for DNA denaturation. Prepare anti-BrdU antibody immediately prior to use, and dilute with incubation buffer at 1:10 to create a working solution. Do not store. (e) Anti-mouse Ig-alkaline phosphatase (AP). Prepare anti- mouse Ig-AP immediately prior to use, and dilute with PBS at 1:10 to create a working solution. Do not store. (f) NBT/BCIP. Prepare NBT/BCIP immediately prior to use. Combine 13 μ L NBT and 10 μ L BCIP solution with 3 mL substrate buffer. This will generate a colored substrate solution. Do not store. 3 Methods 3.1 Immunocyto- 1. Grow limbal stem cells on slides until 50 % confl uent ( see Chapters chemistry for 3 , 7 , and 9 ). Adherent Cells 2. Add BrdU labeling solution to cover the slides and incubate the cells at 37 °C, 5% CO2 . To detect cell proliferation, incubate the cells for 60 min. To detect cell repair, incubate the cells for 30 min ( see Note 1 ). 3. Wash slides three times with diluted washing buffer. 4. Add anti-BrdU antibody working solution to cover the slides and incubate for 30 min at 37 °C ( see Notes 2 and 3 ). 5. Wash slides three times with diluted washing buffer. 6. Add anti-mouse Ig-AP working solution to cover slides and incubate for 30 min at 37 °C. 7. Wash slides three times with diluted washing buffer. 8. Add NBT/BCIP substrate solution to cover the slides and incubate for 30 min at 25 °C. 9. Mount slides and inspect with a bright- fi eld microscope. 68 Ashley M. Crane and Sanjoy K. Bhattacharya 3.2 Immunohisto- Appropriate ethical approval must be obtained before undertaking chemistry for In Vivo research using animals as experimental models. Frozen Tissue Sections 1. Introduce the stock BrdU labeling reagent (2 mL reagent/100 g animal body weight) intravenously into the animal. 2. Sacri fi ce the animal after administering the BrdU labeling reagent. 3. Prepare 5 μ m thick sections of limbus with a cryostat and place on poly- L -lysine glass slides. 4. Fix limbal tissue in ethanol. 5. Wash frozen sections three times with diluted washing buffer and air-dry the area to be stained. 6. Add anti-BrdU antibody working solution to cover the slides and incubate for 30 min at 37 °C. 7. Wash slides three times with diluted washing buffer and air-dry the area to be stained. 8. Add anti-mouse Ig-AP working solution to cover the slides and incubate for 30 min at 37 °C. 9. Wash slides three times with diluted washing buffer and air- dry. 10. Add NBT/BCIP substrate solution to cover the slides and incubate for 30 min at 25 °C. 11. Wash slides three times with diluted washing buffer and air- dry. 12. Mount slides and inspect with a bright- fi eld microscope. 3.3 Immunohisto 1. Cut tissue samples to produce slices that are approximately chemistry for Tissue 1 mm thick. Slices Prepared by 2. Add BrdU labeling solution to cover the cut sample and incu- Frozen Section bate the cells at 37 °C, 5% CO2 . To detect cell proliferation, incubate the cells for 60 min. To detect cell repair, incubate the cells for 30 min. 3. Place the sample in washing buffer and incubate for 30 min at 37 °C. 4. Repeat steps 3 – 12 of Subheading 3.2 . 4 Notes 1. As previously mentioned, the use of BrdU does come with some limitations. The use of BrdU in vivo has been shown to adversely affect cell survival, migration, and fi nal position in the brain [ 3 ] , and may similarly affect limbal stem cells to an unknown extent. Furthermore, with some labeling kits such as The Use of Bromodeoxyuridine Incorporation Assays... 69 BrdU Labeling and Detection Kit II (Roche Diagnostics) there is 10 % cross-reactivity with 5-iodo-2 ¢ deoxy-uridine. 2. The anti-BrdU monoclonal antibody requires DNA denaturation because it is directed against single stranded DNA [ 1 ] . Methods used for denaturation, including heat and nucleases, threaten DNA integrity. Recent new methods of detecting DNA synthesis in dividing cells include the use of another thymidine analog, 5-ethynyl-2¢-deoxyuridine (EdU) and the use of fl uorescent azides and “click” chemistry in in vitro models [9, 10 ] .

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