BD ACCURI NEWS

 

August 2017


Spotlight

Finding new targets for immune-mediated pathologies

Spotlight - Bertin

Samuel Bertin is an assistant project scientist in the Department of Medicine at the University of California, San Diego. His research focuses on identifying new therapeutic targets for immune-mediated pathologies such as inflammatory bowel disease (IBD). Dr. Bertin spoke to us about the role of CD4+ T cells in these diseases, and told us why a BD Accuri™ personal flow cytometer is “quite perfect” for his lab.
Read the interview »

Application Highlight

Using EdU click chemistry to assess cell cycle

Flow cytometry has become an essential methodology for assessing cell cycle, cell proliferation, and DNA content. Multicolor flow cytometric assays allow researchers to investigate these facets of cell status—along with other cellular events, such as apoptosis, DNA damage, protein phosphorylation, or cytokine secretion—within heterogeneous cell populations. With the BD Accuri C6 Plus, you can assess cell cycle and other aspects of cellular activity right on your benchtop.

One standard flow cytometric method for assessing cell cycle employs bromodeoxyuridine (BrdU), an analog of the DNA precursor thymidine. When cells are incubated in the presence of BrdU, the molecule is incorporated into newly synthesized DNA by cells entering and progressing through the S phase of the cell cycle. You can assess the amount of BrdU using a specific flow cytometry antibody against BrdU. To determine the amount of total DNA, samples can also be stained with 7-amino actinomycin D (7-AAD), which binds to DNA.1

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Figure 1. Determining cell cycle phases using BrdU and 7-AAD
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Figure 1. Determining cell cycle phases using BrdU and 7-AAD

Using the BD Pharmingen FITC or APC BrdU Flow Kit, human peripheral blood mononuclear cells (PBMCs) were stimulated, expanded, restimulated and labeled with 20 μM of BrdU during the final hour. After harvesting and staining the cells with 7-AAD and either FITC or APC anti-BrdU according to the kit protocol, samples were acquired on a BD Accuri™ C6 flow cytometer using the kit template, and analyzed using BD Accuri™ C6 software. Cell cycle phases are clearly distinguished in plots showing (A) 7-AAD vs BrdU FITC and (B) 7-AAD vs BrdU APC. Cells in black (to left of the G0/G1 gate) contain less DNA, which may indicate cell death.

With this combination, two-color flow cytometry can determine the cell cycle phase of cells that are actively synthesizing DNA. BrdU assays can assess cell proliferation and apoptosis as well as cell cycle distribution. The whole process generally takes 3–5 hours, including incubation.

Figure 1 shows how antibodies to BrdU and 7-AAD can determine the cell cycle phase of cells that are actively synthesizing DNA. In a dot-plot, the cells form a characteristic horseshoe pattern, as DNA content increases from the G0G1 phase to S to G2/M, and as BrdU is taken up by cells in the S phase. This analysis employed the BD Pharmingen™ FITC and APC BrdU Flow Kits (Cat. Nos. 559619 and 552598), which use BrdU, 7-AAD, and a system of buffers to identify and analyze actively cycling cell subpopulations. BD Accuri™ software templates keyed to each kit can streamline setup and analysis.

BD recently introduced another, more rapid alternative that employs a different thymidine analog, ethynyl deoxyuridine (EdU). This reagent works similarly to BrdU, but is detected by a “click” chemistry reaction between the ethynyl group of EdU and a fluorescent probe containing an azide moiety. In this reaction, the alkyne and azide groups react selectively to produce a fluorescent, covalently linked product that can be detected by flow cytometry. EdU analysis takes fewer preparation steps than BrdU and the whole process can generally be completed in 2–3 hours.

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Figure 2. Determining cell cycle phases using EdU, pHH3 and PI
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Figure 2. Determining cell cycle phases using EdU, pHH3 and PI

MDA-MB-468 breast cancer cells were treated with 100 nM of the microtubule depolymerizing agent nocodazole (Sigma; 1–10,000 nM) for 16 hours. Cells were then pulsed with 10 μM of EdU for 1 hour, harvested from culture, stained for EdU content using the BD Pharmingen 647 EdU Click Proliferation Kit (Cat. No. 565456), subsequently stained with BD Pharmingen Alexa Fluor® 488 Rat anti-Histone H3 (pS28, Cat. No. 558610), and then stained for DNA content with BD Pharmingen Propidium Iodide Staining Solution (Cat. No. 556463) and 0.1 mg/mL of RNase A. Cells were acquired in a 96-well plate using a BD Accuri C6 Plus and BD CSampler Plus, and analyzed using BD Accuri C6 Plus software. Results: In cell cycle analyses, M-phase cells were identified as pHH3+ (left plots). G0/G1, S and G2-phase cells were then discriminated based on EdU and DNA content (right plots), where G0/G1-phase cells are EdU/2N, S-phase cells are EdU+/2N-4N and G2-phase cells are EdU/4N. MDA-MB-468 cells treated with 100 nM of nocodazole (B) showed an increase of cells in the M and G2 phases compared to untreated cells (A).

Figure 2 shows an EdU cell cycle analysis from our recent product information sheet, Automating dose-response cell cycle analysis with the BD CSampler™ Plus. The cells were treated with nocodazole, an antineoplastic agent that interferes with the polymerization of microtubules, causing cells to arrest in the G2 or M phase of the cell cycle. To assess cell cycle, EdU was paired with the DNA dye propidium iodide (PI). The cells were also stained with an antibody against phosphohistone H3 (pHH3) to further differentiate M phase cells from cells in other phases.

The dot plots show cell cycle analyses for a sample of MDA-MB-468 breast cancer cells treated with 100 nM of nocodazole vs untreated cells. First, in the left plot of each condition, pHH3 analysis demonstrated that many treated cells were arrested in the M phase, which showed more than a 10-fold increase over untreated cells. The remaining cells were gated and analyzed (right plots) for EdU vs PI. Again, the cells form a characteristic horseshoe pattern, and the data demonstrated more than a three-fold increase in G2 phase cells, with corresponding decreases in other cell cycle phases.

Both BrdU and EdU kits can be used to generate information about cell cycle status. When time is constrained, the shorter EdU protocol can provide equivalent information more quickly. Please note two other differences between the methods. First, with EdU, antibodies should be stained in a separate step, while with BrdU, antibodies can be stained at the same time as BrdU detection. Second, because the EdU protocol requires the use of copper, multiplexing EdU cell cycle analysis with PE-conjugated antibodies requires protocol optimization due to the quenching of PE by copper. In this case, BrdU flow kits can make it easier to design multiplexed panels.

Download our BD Accuri™ product information sheet on automating dose-response cell cycle analysis »

Download our product information sheet on BD Pharmingen™ proliferation kits for cell cycle analysis »

Download the BD Accuri C6 Plus brochure »


 

Tips & Tricks

Avoid sample clumping

Clumps of sample can clog the fluidics system and interfere with proper operation. To avoid sample clumping, visually inspect each sample prior to acquisition. If cell clumps or chunks of tissue are visible, pass the sample through a 70- or 100-micron cell strainer before running the sample. If you can see it, don't run it.


 

Publication Picks

This section highlights interesting recent articles that describe research using BD Accuri flow cytometers.

Multiple sclerosis therapies

Zhou Y, Leng X, Luo S, et al. Tolerogenic dendritic cells generated with tofacitinib ameliorate experimental autoimmune encephalomyelitis through modulation of Th17/Treg balance. J Immunol Res. 2016;2016:5021537. PubMed

Immune abnormalities

Limgala RP, Ioanou C, Plassmeyer M, et al. Time of initiating enzyme replacement therapy affects immune abnormalities and disease severity in patients with Gaucher disease. PloS One. 2016;11:e0168135. PubMed

Chromosome delivery

Brown DM, Chan YA, Desai PJ, et al. Efficient size-independent chromosome delivery from yeast to cultured cell lines. Nucleic Acids Res. 2017;45:e50. PubMed

Dental sealants

Kim DH, Kwon TY. In vitro study of Streptococcus mutans adhesion on composite resin coated with three surface sealants. Restor Dent Endod. 2017;42:39-47. PubMed

 


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1 All reagents and kits are compatible with both the BD Accuri C6 Plus and the BD Accuri™ C6 flow cytometer systems. Data for Figures 1 and 2 was generated on the BD Accuri C6 and BD Accuri C6 Plus, respectively. Information about BD reagent kits, BD Accuri™ C6 and BD Accuri C6 Plus software templates, and BD CSampler™ and BD CSampler Plus automation options is available at www.bdbiosciences.com/accuri.