Besides monitoring the cell cycle distribution, it is often important to know the proliferative status of a cell population. Protocols in which incorporation of 5-bromo-2'-deoxyuridine (BrdU) that can be subsequently indentified with a labeled antibody (anti-BrdU assay) have been used for the detection of S-phase cells. The two parameter plots, resulting from analysis of DNA content versus the fluorescence of the incorporated anti-BrdU label can allow for simultaneous analysis of cell cycle distribution and identification of cells in DNA synthesis (1–4).
The anti-BrdU assay requires treatment with heat or acid to unmask the binding sites for the anti-BrdU antibody. The type of fixative being used, the degree of DNA denaturation, and non-specific antibody binding are critical factors, which affect the results and some times make the anti-BrdU assay tedious and time consuming (3).
To circumvent some of these problems, a new thymidine analog [5-ethynyl-2'-deoxyuridine (EdU)], which couples with a number of different fluorescent azide dyes via a copper mediated “click” reaction has been recently introduced (5–7). The commercialization of this assay by the Invitrogen Corporation (Carlsbad, California) has made it possible to study cell proliferation without the use of the anti-BrdU antibody. The Invitrogen Click-iT™ EdU Alexa Fluor® 488 Flow Cytometry Assay is based on the use of paraformaldehyde as a fixative and 7-AAD as a DNA binding dye. An essential step in this assay is fixation of the cells with paraformaldehyde, which is known to cause loss of resolution and broadening of G0/G1 peak in the DNA histograms (8, 9). In contrast, ethanol fixation for whole cells or isolated nuclei has been used in the anti-BrdU antibody labeling protocols and can generate DNA distribution histograms with low coefficients of variation (CV's) (1–4). Similarly, in contrast to other DNA binding fluorochromes such as propidium iodide, 7-AAD is known to yield DNA distribution histograms of low resolution with broad CV's (10, 11).
To circumvent some of these problems and improve on the results, we have modified the Click-iT™ EdU Alexa Fluor 488 Flow Cytometry Assay by eliminating the fixation and permeabilization steps, and staining unfixed nuclei isolated by hypotonic lysis with propidium iodide.
MATERIALS AND METHODS
Cell Culture and EdU Labeling
Human leukemic CEM cells in suspension cultures were grown in RPMI 1640 medium supplemented with L-glutamine, 10% bovine calf serum, and antibiotics (penicillin, streptomycin, and Fungizone®). Cells were seeded at 0.5× 106/ml and incubated overnight before harvesting. EdU at a final concentration of 20 μM was added and the cultures incubated at 37°C for 30 min. Aliquots were collected by centrifugation, and the cell pellets washed and resuspended in medium containing 20 μM BrdU as a “cold chase” for the EdU “pulse” experiments. All centrifugations were performed at 550 relative centrifugal force (RCF) for collection of nuclei and at 200 RCF for the whole cells.
Cell Fixation, Permeabilization, and Nuclear Isolation
Cells were fixed for 15 min with paraformaldehyde and permeabilized for 30 min at room temperature in the dark using Triton X-100 solution as per instructions in the Invitrogen kit. For isolation of nuclei, aliquots from suspension cultures incubated with the EdU were centrifuged and the pellets resuspended in 0.5 mL of 0.03% Nonidet P-40 (Igepal CA-630 is an NP-40 equivalent from Sigma-Aldrich, St. Louis, Missouri) in 0.1% sodium citrate for 30 sec (12). Cell suspensions were vortexed and 2.5 mL of Dubecco's phosphate buffered saline containing 1% bovine serum albumin was added.
Click-iT™ and DNA Staining
Fixed cells or unfixed nuclei were stained with the Click-iT™ reaction mixture and 7-AAD with RNase according to the instructions in the assay kit. For propidium iodide staining of the isolated nuclei, we used 4 μg/ml of propidium iodide containing 200 μg/ml of ribonuclease A for 30 min at room temperature in the dark.
Figure 1 shows contour plots (A-C) and DNA histograms (D-F) of cells incubated with 20 μM EdU and stained with the Click-iT™ Assay. Contour plot in Figure 1A and DNA histogram in Figure 1D are of cells fixed with paraformaldehyde and stained with 7-AAD. Histogram in Figure 1D had broad half peak coefficient of variation (HPCV) of the G0/G1 peak (HPCV = 8.46%).
Contour plot in Figure 1B and DNA histogram in Figure 1E are of nuclei isolated by hypotonic lysis and stained with 7-AAD. The HPCV of the G0/G1 peak was 5.8%. In contrast to the paraformaldehyde fixed cells (Figs. 1A and 1D) or the isolated nuclei stained with 7-AAD (Figs. 1B and 1E), the isolated nuclei stained with propidium iodide (Figs. 1C and 1F) had DNA histograms with better resolution (HPCV = 4.62%), and distinct G0/G1 and G2M peaks.
Figure 2 shows EdU labeled cells prepared by the modified isolated nuclei and the propidium iodide staining method. Aliquots were collected 0–6 h after 30 min incubation with EdU, washing and chasing of the label with media containing an excess of BrdU and nuclear isolation with hypotonic citrate/NP-40. These contour plots show the progression of the labeled cells thru the cell cycle. In Figures 2A–2D, a very small population of cells with hyper-tetraploid DNA content is seen in the area beyond the G2M peak.
The method described in this report used suspension cultures of lymphoid cells. To process cells growing in a monolayer, we added EdU label to the flask for 30 min, followed by a wash with saline, and addition of the cell lysis solution to cover the monolayer for 30 sec. The nuclei isolated by this method were processed as a suspension as described eariler.
The processing of unfixed nuclei for anti-BrdU labeling has been described earlier(13). Observations in this study show that fixation with paraformaldehyde and staining with 7-AAD increases the G0/G1 CV, and decreases resolution of the DNA histograms. In contrast, unfixed nuclei stained by propidium iodide had much better resolution. Chart 1 show that staining of the isolated and unfixed nuclei also eliminates the 15 min fixation step, the 30 min permeabilization step, and the centrifugation in between or almost an hour's processing time. In situations, where no cytoplasmic or surface marker staining is required, the modified Click-iT™ method is faster and yields better DNA histograms.