Fig 1.
Optimising the protocol for BrdU detection.
(A) HeLa cells were incubated with BrdU for 30 min, fixed with formaldehyde and BrdU was detected in DNA using 40 mM HCl. BrdU was detected either by the B44 or Bu20a anti-BrdU antibody with exonuclease III. The effect of formaldehyde post-fixation on the signal is shown as well. The data are presented as the mean ± SD. (B-E) A comparison of various HCl concentrations on BrdU signal in HeLa cells labelled for 30 min with BrdU and fixed either with formaldehyde (B, D) or ethanol (C, E). The incorporated BrdU was detected using either B44 (B, C) or Bu20a (D, E) antibody clone with exonuclease III. The data are presented as the mean ± SD. (F, G) A comparison of five monoclonal anti-BrdU antibody clones and one polyclonal antibody is shown. The HeLa cells were labelled with BrdU for 30 min and fixed either with formaldehyde (F) or ethanol (G). The impact of the post-fixation step is shown as well. The data are presented as the mean ± SD. (H) The effect of the length of the washing step on the BrdU-derived signal. The HeLa cells were labelled with BrdU for 30 min and fixed with formaldehyde. After incubation with the primary antibody, the cells were washed for 5 s (0 min) or 5 or 25 min in 1× PBS and then post-fixed with formaldehyde. The data are normalised to the % of the average signal in samples washed for 5 s in 1× PBS and then immediately post-fixed with formaldehyde. The data are presented as the mean ± SD.
Fig 2.
The effect of optimized procedure on the localisation of cellular proteins.
HeLa cells were incubated with BrdU for 30 minutes and fixed with formaldehyde. BrdU was revealed using 20 mM HCl. The proteins SC35, mitochondrial protein MTCO2, histone H1.2 and coilin were concurrently detected with the incorporated BrdU. BrdU was detected using either chicken polyclonal antibody or B44 monoclonal antibody depending on the host producing the antibody for the particular cellular protein. The control cells were not labelled with BrdU and were not treated with HCl and exonuclease III. Proteins are in green, BrdU is in red and DAPI is in blue. Scale bar = 20 μm.
Fig 3.
Fluorescent proteins detection and comparison of replicational signal after short pulses of BrdU and EdU.
(A) The impact of the optimised method on fluorescent proteins is shown. HeLa cells expressing FUCCI were incubated with or without BrdU for 30 minutes and the detection of BrdU using the optimized method was performed. The cells were treated with HCl either for 10 or 20 minutes. The average signal of fluorescent proteins was measured in cells. The data were normalised to the % of the average signal of Fucci-G1 Orange and Fucci-S/G2/M Green in control, non-treated, cells. The data are presented as the mean ± SD. (B) The comparison of the signal in cells labelled with 10 μM EdU or BrdU for 5 minutes. The graphs show differences between cells incubated either with EdU or BrdU and fixed either with formaldehyde or ethanol. The data are presented as the mean ± SD. (C, D) The histograms of the mean nuclear EdU- (C) or BrdU-derived (D) signals in formaldehyde-fixed cells labelled with 10 μM EdU or BrdU for 5 minutes. (E, F) The histograms of the mean nuclear EdU- (E) or BrdU-derived (F) signals in ethanol-fixed cells labelled with 10 μM BrdU or EdU for 5 minutes.
Fig 4.
Fluorescence images of BrdU, EdU and TFdU.
(A, B) The detection of EdU and BrdU in HeLa cells labelled with EdU or BrdU for 5 minutes and fixed with formaldehyde (A) or ethanol (B). EdU and BrdU are in green, DAPI is in blue. The scale bar—50 μm. (C) The detection of TFdU. HeLa cells were labelled with TFdU for 30 min. TFdU was revealed by optimized method and detected by anti-BrdU antibody clone 3D4. TFdU is in green, DAPI is in blue. The scale bar—50 μm.
Fig 5.
Determination of cell cycle by image and flow cytometry.
(A) The bivariate analysis of the replication signal and DNA content by DAPI in HeLa cells after a 30-minute incubation with 10 μM BrdU using image cytometry. (B) The bivariate analysis of the replication signal and DNA content by propidium iodide in A549 cells after a 30-minute incubation with 10 μM BrdU using flow cytometry.