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EdU (5-ethynyl-2′-deoxyuridine) incorporation has proved advantageous in the studies of cell kinetics, DNA synthesis, and cellular proliferation in vitro and in vivo compared to [3H]thymidine incorporation and BrdU (5-bromo-2′-deoxyuridine) incorporation. Here, we describe a method that combines EdU incorporation and immunostaining with flow cytometric analysis to detect the proliferations of T lymphocyte subsets in vitro and optimized the assay's conditions. We found that the number of EdU+ cells were associated with EdU concentration, incubation time, and the volume of Click reaction solution, the best EdU concentration 10–50 μM, the optimal incubation time 8–12 h and the proper volume of Click volume 100 μl for labeling 1 × 106 lymphocytes. Fixation was better to be performed before permeabilization, not together with. Furthermore, the permeabilization detergent reagent, PBS with 0.05% saponin was better than Tris buffer saline (TBS) with 0.1% Triton X-100. In addition, sufficient wash with PBS with 0.05% saponin has no influence on the staining of EdU+ cells. Also, the lymphocytes incorporating EdU could be stored at 4°C, −80°C, and in liquid nitrogen up to 21 days. The present study will aid in optimization of flow cytometry assay to detect the proliferations of T cell subsets by EdU incorporation and the labeling of cell surface antigens. © 2012 International Society for Advancement of Cytometry
The common assays for detecting DNA synthesis in proliferation cells are [3H] thymidine and BrdU incorporation (1). The traditional method of [3H]thymidine incorporated into DNA is detected by autoradiography, which is cumbersome and time-consuming. BrdU immunostaining method is a more convenient approach with simplified procedures and high efficiency. However, the major impediment of BrdU immunostaining is the complementary bases pairing in double-stranded DNA, which blocks the binding of the anti-BrdU antibody to BrdU. Hence, strong denaturing conditions such as digestion with DNase or concentrated hydrochloric acid have to be applied for the exposure of BrdU epitopes. So the intensity of BrdU staining highly depends on the conditions (2). Therefore, a simple and rapid nucleic acid labeling technique with considerable sensitivity and stability is still needed for the studies of cell kinetics, DNA synthesis, and cellular proliferation in vitro and in vivo. In particular, techniques which are environmentally friendly and do not destroy the ultrastructure of sample are highly desirable. The recently developed EdU incorporation (3) overcomes the disadvantages mentioned above, and can thus be used to label the replicated DNA in the context of well preserved cellular and chromatin ultrastructure. As a thymidine analog, EdU is used by replacing the methyl group at position 5 of deoxyuridine with an alkyne group. It is reported that EdU is readily incorporated into cellular DNA during the S phase of the cell cycle as shown in NIH 3T3 cells (3), neuron (4, 5), Xenopus egg extracts (3), human T-cell leukemia Jurkat cell line (6), human breast cancer cell lines SK-BR-3 and BT474 (7), murine mesenchymal stem cells (8), mouse epithelial cells (9) as well as the spleen cells and the lymph node cells of mouse (10, 11). The terminal alkyne group of EdU can react with fluorescent azides in a Cu(I)-catalyzed [3+2] cycloaddition (“click” chemistry). This reaction enables detection of EdU incorporation into cells by fluorescent microscopy or fluorescence activated cell sorter. Recently, EdU incorporation combined with fluorescent microscopy is widely used in vitro and in vivo. However, few groups have used flow cytometry analysis of EdU incorporation and cell surface antigens in evaluating the proliferations of mouse T lymphocyte subsets (10). In their study, multiple cell surface antibodies simultaneously labeling T cells was not mentioned and the factors affecting the entire assay were not fully investigated. Moreover, the manufacturer's suggested protocols (Cell-Light™ EdU Cell Proliferation Detection from RiBoBio, Guangzhou, China) often result in a significantly weakened fluorescence intensity of cell surface antibodies such as CD3e-PerCP-Cy5.5, CD4-FITC, CD8a-PE. Hence, we investigated the effect of reagents and applications related to polychromatic flow cytometry in this assay.
Finally, we propose a feasible protocol of flow cytometry assay to detect the proliferations of T cell subsets by EdU incorporation and the labeling of cell surface antigens.
- Top of page
- MATERIALS AND METHODS
- Literature Cited
- Supporting Information
Although flow cytometry analysis of the proliferations of T lymphocytes subsets, which was performed through labeling of cell surface antigens combined with EdU incorporation, has been used to measure T-cell proliferation in vivo (8, 11), it has not been well established to detect T-cell proliferation in vitro. Therefore, we tried to optimize the conditions for this assay in vitro and to establish a feasible and accurate protocol for the detection of T-cell proliferation.
A few general conclusions could be drawn from this study. First, the number of EdU+ cells was associated with EdU concentration, EdU incubation time and the volume of Click reaction solution. We found that 10–50 μM EdU efficiently labeled T cells. However, the increasing EdU concentrations from 150 to 250 μM caused significant decrease of the labeled cells, which further proved the suppression of EdU on cell proliferation (10, 14, 15). Also, the optimal EdU incubation time was determined, which was from 8 to 12 h. As to the volume of Click reaction solution, 100 μl, 1/5 of recommended volume in two manufacturers' protocols, was well suited for the staining of initiating cells to 1 × 106. Considering that fixative may induce change in antibody binding capacity because of potential alteration on epitope exposure and affinity (16), Click reaction solution was recommended to be added before fixation. Besides, after Click reaction, sufficient wash was helpful for the reduction of the nonspecific fluorescence intensity of Apollo® 643 azide, but had no effect on the proportion of EdU+ cells.
Second, fixation was implicated to play a pivotal role in maintaining the cell morphology and the fluorescence intensity of cell surface antibodies, which varied with cell types and surface markers (17); therefore, a proper fixation for lymphocytes was important. Our results revealed that this step was better to be performed before permeabilization, and the suitable incubation time was 15 minutes for lymphocytes.
Third, for permeabilization the detergent reagent saponin was milder than Triton X-100 which could lyse cell membrane more thoroughly; this result was similar to the reports in primary adult epidermal keratinocytes (9). Furthermore, saponin not only lysed the cell membrane properly, but also maintained the fluorescence intensity of cell surface antibodies, which was consistent with the previous observations (9, 13, 18).
Fourth, strong correlation between EdU and [3H]thymidine incorporation indicated EdU incorporation was as accurate as [3H]thymidine incorporation to detect PHA stimulated murine T lymphocytes proliferation; this was similar to previous report on anti-CD3 stimulated murine T lymphocytes (10).
Finally, EdU labeled cells could be stored at 4°C, −80°C or in liquid nitrogen for storage periods up to 21 days, with the stable proportions of EdU+ cells in CD4+ T cells and surface staining in lymphocytes.
In summary, we described here an optimized technology of EdU incorporation and the labeling of cell surface antigens combined with flow cytometry analysis (Table 1), which could be used to detect the proliferations of T lymphocyte subsets accurately. Different factors contribute to this method include EdU concentration, EdU incubation time, fixation, permeabilization, Click reaction, wash reagents, extent of wash, which may have significant effects on final results; therefore, these factors should be scrutinized in immunofluorescence experiments. This method does not require strong denaturing conditions, anti-BrdU antibodies, and radioactive substance as required in the aforementioned methods. However, there are still some limitations in this method compared with BrdU incorporation and [3H]thymidine incorporation. For example, the cost of EdU incorporation is higher than that of [3H]thymidine incorporation (10). The present study not only can aid in the optimization of flow cytometry staining panels to quantify T-cell proliferation in mouse, but also can serve as a particular protocol of T-cell proliferation detection by the labeling of cell surface antigens combined with EdU incorporation.
Table 1. The protocol for flow cytometry analysis of the proliferations of T-lymphocyte subsets by the labeling of cell surface antigens combined with EdU incorporation
|1||Isolation and culture of lymphocytes|
|3||Immunofluorescence staining of cell surface antigens|
|4||Fixation and permeabilization|