Dual Pulse Labeling of Cell Proliferation—Combining Click Chemistry with Highly Specific BrdU Antibodies

 

The measurement of cell proliferation is fundamental to the assessment of cell health, genotoxicity, and drug efficacy. Proliferation is traditionally assessed by incubating cells with a single “pulse” of a nucleoside analog that is incorporated into DNA and detected using radioactivity, antibodies, or click chemistry. Some applications, such as drug efficacy testing, benefit from the incorporation of two different analogs at different time points (dual pulse labeling), which can further define cell cycle kinetics. With the availability of new highly specific anti-BrdU antibodies, the BrdU labeling technique can be combined with click chemistry detection for a simplified method of dual pulse labeling.

 

Combining the Benefits of EdU and BrdU

Traditionally, the detection of cell proliferation has employed the incorporation of the thymidine analog BrdU (5-bromo-2´-deoxyuridine) during DNA synthesis, followed by detection with an anti-BrdU antibody [1–4]. This method is rapidly being replaced by the click chemistry–based Click-iT® EdU assay [5,6], because unlike BrdU assays, Click-iT® EdU assays are not antibody-based and therefore do not require DNA denaturation for detection of the incorporated nucleoside. Click-iT® assays use a modified nucleoside, EdU (5-ethynyl-2´-deoxyuridine), that is incorporated during DNA synthesis and detected using a click reaction—a copper(I)-catalyzed reaction between an azide and an alkyne.

How Click-iT® EdU Works


Click-iT® EdU – click chemistry simplicity makes it the faster, friendlier alternative to 3H-thymidine and BrdU. Unlike assays using bromodeoxyuridine (BrdU), Click-iT® EdU assays are not antibody based and therefore do not require DNA denaturation for detection of the incorporated nucleoside. Instead, Click-iT® EdU utilizes click chemistry for detection in a variety of Alexa Fluor® dye fluorescent readouts. Furthermore, the streamlined detection protocol both reduces the total number of steps and significantly decreases the total amount of time.

Highly Specific Detection of EdU and BrdU

Dual pulse labeling of cell proliferation traditionally uses BrdU immunocytochemistry with iododeoxyuridine (IdU) or chlorodeoxyuridine (CldU), using multiple BrdU antibodies of different clones that cross-react with IdU and CldU for detection [7]. This complex method can be greatly simplified by using sequential pulses of the thymidine analogs EdU and BrdU, but a major challenge is that many anti-BrdU antibodies exhibit cross-reactivity with EdU.

The new anti-BrdU antibody clone MoBU-1 displays no cross-reactivity with EdU used in the Click-iT® EdU assay (Figure 1). Detection with this dual pulse method is highly specific for each modified nucleoside and does not require media replacement or removal of EdU before the addition of BrdU (Figure 2).

Anti-BrDU Does Not React With EdU

Figure 1. The anti-BrdU antibody clone MoBU-1 does not react with EdU. Jurkat T cells were treated with 10 μM EdU for 1 hr, the cells were fixed in ethanol, and an acid denaturation method was used before labeling with FITC conjugates of the most commonly used anti-BrdU clones: 3D4, PRB-1, and MoBU-1. Cells were analyzed with a BD™ LSR II flow cytometer using a 488 nm excitation laser, and fluorescence was collected with a 530/30 bandpass filter. Of the clones tested, only clone MoBU-1 did not react with EdU.
Dual Pulse Labeling of Cell Proliferation with BrdU and Click-iT® EdU

Figure 2. Dual pulse labeling of cell proliferation with BrdU and Click-iT® EdU.TF-1 erythroblast cells were pulsed with 20 μM EdU for 1 hr followed by 10 μM BrdU for 1 hr. The cells were fixed in ethanol, and an acid denaturation method was used before labeling with anti-BrdU (clone MoBU-1)–Alexa Fluor® 488 conjugate and Click-iT® EdU–Alexa Fluor® 647 azide. Data were collected with a BD™ LSR II flow cytometer using 488 nm excitation with a 530/30 bandpass filter, and 633 nm excitation with a 660/20 bandpass filter. Cells colored blue are negative for both EdU and BrdU; cells colored dark green are positive for both EdU and BrdU; cells colored red are positive for EdU but negative for BrdU; cells colored light green are negative for EdU but positive for BrdU.

A Simple Assay for a Diverse Set of Applications

The MoBU-1 anti-BrdU antibody clone has high specificity for BrdU in addition to having no cross-reactivity with EdU. This clone can also readily detect bromouridine (BrU) incorporated into RNA or BrdUTP incorporated into DNA strand breaks via the TUNEL assay. Conjugates of MoBU-1 can be directly measured by flow cytometry, fluorescence microscopy, and high-content imaging (Figure 3). The unconjugated form and the biotin conjugate of this clone require a secondary antibody or a streptavidin conjugate for detection, respectively.
Dual pulse labeling of cell proliferation using high-content imaging<br>and analysis.

Figure 3. Dual pulse labeling of cell proliferation using high-content imaging and analysis. The effect of various drugs on DNA replication was assessed using dual pulse labeling. U2OS cells were pulsed with 10 μM EdU for 60 min, washed, then treated with chloroquine, rosiglitazone, acetaminophen, or etoposide, or left untreated for 23 hr. Cells were pulsed with 10 μM BrdU for 60 min, then fixed and denatured before performing the Click-iT® EdU Alexa Fluor® 488 Imaging Assay to detect EdU (green). BrdU incorporation was detected using anti-BrdU (clone MoBU-1)–Alexa Fluor® 594 conjugate (red). Nuclei were counterstained with HCS NuclearMask™ Blue stain. (Left) Control cells dual-labeled with EdU and BrdU, imaged with a Zeiss Axiovert® 200M microscope. (Right) DNA replication was decreased by several drugs, as indicated by a decrease in BrdU-positive cells compared to EdU-positive cells. Data were acquired using the Arrayscan® VTI platform (Thermo Scientific Cellomics®).

References

  1. Gratzner HG (1982) Science 218:474–475.
  2. Dolbeare F, Selden JR (1994) Methods Cell Biol 41:297–316.
  3. Li X, Darzynkiewicz Z (1995) Cell Prolif 28:571–579.
  4. Conboy MJ, Karasov AO, Rando TA (2007) PLoS Biol 5:1120–1126.
  5. Salic A, Mitchison TJ (2008) Proc Natl Acad Sci U S A 105:2415–2420.
  6. Buck SB, Bradford J, Gee KR et al. (2008) Biotechniques 44:927–929.
  7. Current Protocols in Cytometry Vol. 1, JP Robinson, Ed., John Wiley & Sons Inc. (2007).
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