RNA polymerase; DNA dye

7-Aminoactinomycin D (7-AAD) is a DNA dye that may differentiate between living cells, liver cancer cells, and late-staining/dead cells in flow cytometry. 7-Aminoactinomycin D staining at doses of 5 μg/mL, 10 μg/mL and 20 μg/mL instead of 1 μg/mL is acceptable for measurement of cell filling in flow cytometry [1]. 7-Aminoactinomycin D is typically used at low concentrations (0.5-5 μg/mL) for labeling and elimination of dead cells during flow operations. At higher concentrations (10 -20 μg/mL), 7-aminoactinomycin D has also been used to differentiate between live cells (7-AAD negative) and scanned cells (7-AADdim) or dead cells (7-AADbright ), according to the permeability of the cell membrane and the fluorescence intensity, it is low in early silicon cells and high in late silicon cells and dead cells [1].

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7-Aminoactinomycin D (7-AAD) is a fluorescent derivative of actinomycin D that selectively binds to GC regions of the DNA. 7-AAD is frequently used to stain and exclude dead cells in flow cytometry at low concentrations (0.5–5 μg/ml). At higher concentrations (10–20 μg/ml), 7-AAD has also been used to distinguish between viable cells (7-AADnegative) and apoptotic cells (7-AADdim) or dead cells (7-AADbright) using the fact that permeability of the cell membrane, and hence fluorescence intensity, is low in early apoptotic cells and high in late apoptotic and dead cells. 7-AAD successfully labels cells during the early phase of apoptosis to a degree comparable to annexin V, which binds to extracellular phosphatidylserine of early apoptotic cells. As demonstrated by cell sorting and subsequent TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay and morphological assessment, staining of Jurkat and T3M-4 cells with 20 μg/ml 7-AAD for 20 min at 4 °C correctly identifies different apoptotic populations. Successful staining with 7-Aminoactinomycin D (7-AAD) has also been reported with 10 μg/ml for 30 min, but optimal concentrations for the quantification of apoptosis have not been assessed. This is a major drawback and could be the reason for the rare application of this simple and cheap one-color staining method that can easily be combined with antibody or functional staining. A further disadvantage of the available protocols is a time-consuming fixation step. Therefore, we simplified the protocol, removed the fixation step, and tested several 7-AAD concentrations (1–20 μg/ml) in peripheral blood mononuclear cells (PBMCs) and human leukemia (CEM) cells. The aim of this study was to analyze the suitability of several 7-AAD concentrations for the quantification of apoptosis in flow cytometry [2].

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The potent RNA polymerase inhibitors actinomycin D and 7-Aminoactinomycin D (7-AAD) are shown to bind to single-stranded DNAs. The binding occurs with particular DNA sequences containing guanine residues and is characterized by hypochromic UV absorption changes similar to those observed in interactions of the drugs with double-stranded duplex DNAs. The most striking feature of the binding is the dramatic (ca. 37-fold) enhancement in fluorescence that occurs when the 7-aminoactinomycin is bound to certain single-stranded DNAs. This fluorescence of the complex is also characterized by a 40-nm hypsochromic shift in the emission spectrum of the drug and an increase in the emission anisotropy relative to the free drug or the drug bound to calf thymus DNA. The fluorescence lifetimes change in the presence of the single-stranded DNA in a manner compatible with the intensity difference. Thus, there is an increase in the fraction of the emission corresponding to a 2-ns lifetime component compared to the predominant approximately 0.5-ns lifetime of the free drug. The 7-aminoactinomycin D comigrates in polyacrylamide gels with the single-stranded DNAs, and the fluorescence of the bound drug can be visualized by excitation with 540-nm light. The binding interactions are characterized by association constants of 2.0 x 10(6) to 1.1 x 10(7) M-1 [1].

PBMCs were isolated from fresh heparinized blood of healthy donors, who gave written informed consent, by ficoll density gradient centrifugation. PBMCs were washed twice with phosphate-buffered saline and frozen in fetal calf serum supplemented with 10% dimethyl sulfoxide until analysis. CEM cells were cultivated under standard cell culture conditions. To compare several 7-Aminoactinomycin D (7-AAD) staining protocols, PBMCs were treated with 100 nM staurosporine for 24 h as described previously with modifications and subsequently stained with 7-Aminoactinomycin D (7-AAD) or with the BD Pharmingen PE–Annexin V Apoptosis Detection Kit I (hereafter PE–annexin detection kit), which contains annexin V conjugated to phycoerythrin (PE) and 7-AAD for the detection of apoptotic and dead cells, respectively. The kit was used according to the manufacturer’s instructions. 7-AAD staining was conducted as described previously with minor modifications but without fixation. In brief, treated and untreated (background control) PBMCs (105 cells/sample) were washed with RPMI 1640 cell culture medium (Invitrogen, Karlsruhe, Germany) supplemented with 2% FCS (37 °C) and centrifuged for 5 min at 400g. Each sample was resuspended in 500 μl of RPMI with 2% FCS. Then 7-AAD (stock solution 1 mg in 50 μl methanol and 950 μl PBS with 0.9 mM Ca2+ and 0.5 mM Mg2+) was added to obtain concentrations of 1, 5, 10, and 20 μg/ml. Samples were incubated for 20 min on ice in darkness (n = 3–9). After incubation, all samples were centrifuged (400g, 5 min, 4 °C), washed once with 1 ml of PBS with 2% FCS (4 °C), centrifuged again (400g, 5 min, 4 °C), and resuspended in 500 μl of PBS with 2% FCS (4 °C). Samples were stored on ice and analyzed by flow cytometry within 1 h on a BD LSRII flow cytometer equipped with FACS Diva 6.0 software. 7-AAD and PE were excited at 488 nm. Fluorescence of PE was analyzed at 575/26 nm, and fluorescence of 7-AAD was analyzed at 695/40 nm. PE and 7-AAD were compensated according to the manufacturer’s instructions. Data were further analyzed with FlowJo 7.6.5. Lymphocytes and CEM cells were gated in the forward scatter versus side scatter dot plot according to size and granularity. Viable, apoptotic, and late apoptotic/dead cells were analyzed in a forward scatter versus 7-AAD dot plot. Two control samples were used for standardized gating of cell populations (Fig. 1). Untreated and unstained cells served as a control for viable cells. To accurately and reproducibly set the gates of apoptotic and late apoptotic/dead cells, we measured a second control sample that was stained with 1, 5, 10, or 20 μg/ml 7-AAD and saponine at a concentration of 0.01% (PBMCs) or 0.08% (CEM cells). Saponines as surface-active glycosides permeabilize cellular membranes, thereby imitating leakiness of late apoptotic/dead cells. The gate defined by saponine-treated cells represented late apoptotic/dead cells (Fig. 1). Fluorescence intensity values between late apoptotic/dead and viable cells were considered to represent apoptotic cells. Statistical significance of differences between staining protocols was evaluated by one-way analysis of variance (ANOVA) with GraphPad InStat version 3.10。 Fig. 2A shows the percentages of apoptotic and late apoptotic/dead cells of untreated lymphocytes and lymphocytes treated with staurosporine for 24 h. The absolute percentages of viable cells stained by the PE–annexin detection kit are roughly 10% lower than the percentages in cells stained with 7-AAD. The percentages of viable cells in untreated samples differed significantly between the PE–annexin detection kit and 1 μg/ml (P < 0.001), 5 μg/ml (P < 0.001), 10 μg/ml (P < 0.001), and 20 μg/ml (P < 0.01) 7-AAD stained samples. Because all samples were treated identically except for different staining protocols, the background values of apoptosis were expected to be similar. The underlying reason is most likely that the described staining procedures themselves vary in their influence on the viability of the cells. In consideration of this fact, it is important to always subtract background controls when analyzing the potency of apoptosis-inducing agents. For further analysis, therefore, we analyzed the alteration of apoptosis in treated cells to untreated cells as a ratio (treated divided by untreated) for viable cells and analyzed the difference (treated minus untreated) for apoptotic and late apoptotic/dead cells as percentages of total cell counts. The ratios of viable lymphocytes were 0.91 ± 0.03 (mean ± standard deviation) (1 μg/ml, 20 min), 0.87 ± 0.03 (5 μg/ml, 20 min), 0.87 ± 0.04 (10 μg/ml, 20 min), 0.83 ± 0.07 (20 μg/ml, 20 min), and 0.80 ± 0.1 (PE–annexin detection kit). Relative frequencies of apoptotic and late apoptotic/dead cells are shown in Fig. 2B. Coefficients of variation were 0.46 (1 μg/ml, 20 min), 0.37 (5 μg/ml, 20 min), 0.34 (10 μg/ml, 20 min), 0.57 (20 μg/ml, 20 min), and 0.47 (PE–annexin detection kit). Across a concentration range of 5 to 20 μg/ml, 7-AAD staining revealed rather similar results well in line with the results of the widely accepted commercially available PE–annexin detection kit. One-way ANOVA with Bonferroni post hoc test revealed that the difference of means between 1 μg/ml 7-AAD and the PE–annexin detection kit was significant (P < 0.01), whereas the other staining methods did not differ significantly. Because 5, 10, and 20 μg/ml 7-AAD appeared to be equally suitable for apoptosis staining in flow cytometry, we chose to characterize the most economic protocol using 5 μg/ml in more detail. Therefore, we applied this protocol to a second cell line (human leukemia CEM cells), which is easier to obtain than PBMCs, and compared it for further characterization with the PE–annexin detection kit and the Cell Death Detection ELISAPLUS (hereafter ELISA [enzyme-linked immunosorbent assay] detection kit), a cytometry-independent method that is based on immunochemical determination of histone-complexed DNA fragments. CEM cells were treated with 11.5 μM camptothecin for 16 h (n = 4) or with 150 μM vincristine for 24 h (n = 4). Untreated CEM cells served as background control. 7-AAD staining was conducted as described above with the minor modification that CEM cells were resuspended in 250 μl of RPMI 2% FCS containing 5 μg/ml 7-AAD that was freshly prepared in one solution for all samples. The ELISA detection kit was conducted according to the manufacturer’s instructions (n = 8). The test quantifies apoptosis as the fold increase in the level of apoptosis in treated samples to untreated samples. Because calculation of percentage differences of apoptotic cells is not possible, we deviated from the manufacturer’s instructions for the analysis of the PE–annexin detection kit. As described previously for the comparison of other staining methods, we calculated the fold increase of apoptosis by dividing the percentage of treated apoptotic CEM cells by the percentage of untreated apoptotic CEM cells to be able to compare the results of all three assays. The ratios of apoptosis were 13.8 ± 1.1 (5 μg/ml 7-AAD), 11.1 ± 1.8 (PE–annexin detection kit), and 8.0 ± 4.0 (ELISA detection kit) in camptothecin-treated CEM cells. The coefficients of variation were 0.08, 0.16, and 0.5, respectively. Comparison of the ratio of apoptosis for the different methods (evaluated by one-way ANOVA with Bonferroni post hoc test) demonstrated a significant difference only for 7-AAD versus the ELISA detection kit (P < 0.05) in camptothecin-treated cells. The ratios in vincristine-treated CEM cells were 4.0 ± 0.6 (5 μg/ml 7-AAD, 20 min), 1.3 ± 0.1 (PE–annexin detection kit), and 5.7 ± 0.5 (ELISA detection kit). The coefficients of variation were 0.02, 0.08, and 0.09, respectively. The ratio of apoptosis differed significantly between the methods (7-AAD vs. PE–annexin detection kit and PE–annexin detection kit vs. ELISA detection kit, P < 0.001, and 7-AAD vs. ELISA detection kit, P < 0.01). These results revealed that the three assays give different results depending on the apoptosis-inducing agent. The underlying reason is most likely that the described methods are based on different biochemical mechanisms involved in apoptosis. 7-AAD is a fluorescent DNA dye that can pass the plasma membrane only of apoptotic or dead cells, annexin V binds to extracellular phosphatidylserine, and the ELISA detection kit immunochemically determines histone-complexed DNA fragments of apoptotic cells. Application of the assays, however, is not restrained if this fact is taken into account when interpreting results. Because apoptosis detection assays themselves could influence the absolute percentages of apoptotic cells, it is further important to always analyze results in relation to untreated controls [1].

[1]. Actinomycin D and 7-aminoactinomycin D binding to single-stranded DNA. Biochemistry. 1991 Oct 1;30(39):9469-78.

[2]. 7-Aminoactinomycin D for apoptosis staining in flow cytometry. Anal Biochem. 2012 Oct 1;429(1):79-81.

[3]. Molecular detection of Chlamydia trachomatis and other sexually transmitted bacteria in semen of male partners of infertile couples in Tunisia: the effect on semen parameters and spermatozoa apoptosis markers. PLoS One. 2014 Jul 14;9(7):e98903.

7-AAD is a cyclodepsipeptide.
7-Aminoactinomycin D is a fluorescent nucleic acid dye which selectively binds GC sequences in double-stranded DNA. It has a molecular weight of 1270.5, an absorbance maximun at 546 nm, and emission maximum at 647 nm. It is commonly used to discriminate viable from non-viable cells.
See also: 7-Aminoactinomycin D (annotation moved to).

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7-Aminoactinomycin D (7-AAD) is a DNA dye that distinguishes viable, apoptotic, and late apoptotic/dead cells in flow cytometry. Several staining protocols using 7-AAD have been described, but data on the influence of the 7-AAD concentration on the readout are not available. Therefore, we compared the results obtained by staining with 1, 5, 10, and 20 μg/ml 7-AAD for 20 min with the PE–Annexin V Apoptosis Detection Kit and Cell Death Detection ELISAPLUS in lymphocytes and CEM human leukemia cells. The results showed that 7-AAD staining with 5, 10, and 20 μg/ml, but not with 1 μg/ml, is suitable for quantification of apoptosis in flow cytometry.[2]

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It has been shown previously by cell sorting and subsequent TUNEL assay and morphological assessment staining of Jurkat and T3M-4 cells with 20 μg/ml 7-AAD for 20 min at 4 °C that 7-AAD correctly identifies early and late apoptotic/dead cell populations and that it can be used for analysis of apoptosis by flow cytometry. Successful staining with 7-AAD has also been reported with 10 μg/ml for 30 min. The current study, for the first time, compared the influence of different 7-AAD concentrations on the readout of the assay. We could demonstrate, for the first time, that 7-AAD staining with 5, 10, and 20 μg/ml (20 min, 4 °C, in darkness) was equally suitable for the staining of early apoptotic and late apoptotic/dead lymphocytes and CEM cells, whereas 1 μg/ml should not be used. Moreover, we improved the protocol by omitting the time-consuming fixation step. Taken together, the 7-AAD assay is a cheap and simple method for quantification of apoptosis. [2]

C62H87N13O16

1270.43168

1269.639

C, 58.62; H, 6.90; N, 14.33; O, 20.15

7240-37-1

65180

Brown to reddish brown solid powder

1.4±0.1 g/cm3

1400.8±65.0 °C at 760 mmHg

801.0±34.3 °C

0.0±0.3 mmHg at 25°C

1.664

-5.02

6

19

8

91

3070

0

O=C(N[C@H](C(N1[C@@](C(N(C)CC(N(C)[C@@H](C(C)C)C(O[C@@H]2C)=O)=O)=O)([H])CCC1)=O)C(C)C)[C@H]2NC(C(C(C(OC3=C(C)C(N)=C4)=C5C)=NC3=C4C(N[C@@H]6C(N[C@H](C(N7[C@@](C(N(C)CC(N(C)[C@@H](C(C)C)C(O[C@@H]6C)=O)=O)=O)([H])CCC7)=O)C(C)C)=O)=O)=C(N)C5=O)=O

YXHLJMWYDTXDHS-UHFFFAOYSA-N

InChI=1S/C62H87N13O16/c1-26(2)42-59(85)74-21-17-19-36(74)57(83)70(13)24-38(76)72(15)48(28(5)6)61(87)89-32(11)44(55(81)66-42)68-53(79)34-23-35(63)30(9)51-46(34)65-47-40(41(64)50(78)31(10)52(47)91-51)54(80)69-45-33(12)90-62(88)49(29(7)8)73(16)39(77)25-71(14)58(84)37-20-18-22-75(37)60(86)43(27(3)4)67-56(45)82/h23,26-29,32-33,36-37,42-45,48-49H,17-22,24-25,63-64H2,1-16H3,(H,66,81)(H,67,82)(H,68,79)(H,69,80)

2,7-diamino-4,6-dimethyl-3-oxo-1-N,9-N-bis[7,11,14-trimethyl-2,5,9,12,15-pentaoxo-3,10-di(propan-2-yl)-8-oxa-1,4,11,14-tetrazabicyclo[14.3.0]nonadecan-6-yl]phenoxazine-1,9-dicarboxamide

7-Aminoactinomycin D; 7240-37-1; Actinomycin D, 7-amino-; FLU 402; 7-amino-AMD; 7-AAD; 7-Amino-actinomycin D; 7-Amino Actinomycin D;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). This product is not stable in solution, please use freshly prepared working solution for optimal results.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~78.71 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)