Apo direct

Manufactured by BD

Sourced in United States

Apo-Direct is a laboratory instrument that measures apoptosis, a process of programmed cell death. The core function of Apo-Direct is to quantify the extent of apoptosis in cell samples using flow cytometry analysis.

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8 protocols using apo direct

Evaluating Sperm Oxidative Stress and DNA Integrity

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ROS formation was measured by chemiluminescence assay with luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) as the probe, using an AutoLumat LB 953 multi-tube luminometer (Berthold Technologies, Oak Ridge, TN, USA). Results were expressed as relative light units RLU/s/10⁶ sperm.25 (link) Total antioxidant capacity (TAC) was measured in the seminal plasma using the antioxidant assay kit (Cayman Chemical, Ann Arbor, MI, USA). Trolox standards and reagent were prepared as per the manufacturer's instructions at the time of the assay. Absorbance was monitored at 750 nm using ELx800 Absorbance Microplate Reader. Results were expressed as micromoles of Trolox.21 (link)
Sperm DNA fragmentation was evaluated using a terminal deoxynucleotidyl transferase–mediated fluorescein–dUTP nick end labeling (TUNEL) assay with an apoptosis-detection kit (Apo-Direct, BD Biosciences Pharmingen, San Diego, CA, USA) and flow cytometry. All fluorescence signals of labeled spermatozoa were analyzed by flow cytometer. Totally, 10 000 spermatozoa were examined, and the percentage of TUNEL-positive cells was calculated.26 (link)

Agarwal A., Sharma R., Samanta L., Durairajanayagam D, & Sabanegh E. (2015). Proteomic signatures of infertile men with clinical varicocele and their validation studies reveal mitochondrial dysfunction leading to infertility. Asian Journal of Andrology, 18(2), 282-291.

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Quantification of Apoptotic Cells by Flow Cytometry

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TUNEL positive cells were quantified using APO-DIRECT™ assay [terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling (TUNEL)] from BD Pharmingen (BD Biosciences, San Jose, CA). Samples were analyzed by using a FC-500 flow cytometer (Beckman Coulter, Inc., Irving, TX) as previously described (20 (link), 21 (link)). All experiments were repeated at least three times with similar results each time.

Li L.S., Reddy S., Lin Z.H., Liu S., Park H., Chun S.G., Bornmann W.G., Thibodeaux J., Yan J., Chakrabarti G., Xie X.J., Sumer B.D., Boothman D.A, & Yordy J.S. (2016). NQO1-mediated tumor-selective lethality and radiosensitization for head and neck cancer. Molecular cancer therapeutics, 15(7), 1757-1767.

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Quantifying Sperm DNA Damage via TUNEL Assay

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Sperm DNA damage was quantified using the terminal deoxynucleotidyl transferase (TdT)-mediated fluorescein-dUTP nick end labeling (TUNEL) assay kit (Apo-Direct; Pharmingen, San Diego, CA) [22 (link)]. This is based on the principal that TdT binds to the 3′-hydroxyl (OH) termini of the single and double strand DNA breaks. It is labeled with, fluorescein isothiocyanate tagged 2′-deoxyuridine, 5′-triphosphate nucleotides (FITC-dUTP) followed by flow cytometry. Briefly, after fixation with 3.7% paraformaldehyde for 30 minutes on ice, spermatozoa were washed and resuspended in ice cold 70% ethanol and then in phosphate buffered saline. The specimens were centrifuged at 1600 rpm for 7 minutes, and the pellet was resuspended for 60 minutes at 37°C in 50 μL of a staining solution containing terminal deoxynucleotidyl transferase (TdT) enzyme, TdT reaction buffer, FITC-dUTP and distilled water. Both negative (without TdT) and positive samples (DNase I) were included. After centrifugation, the cells were washed twice in rinse buffer, resuspended in 0.5 mL of propidium iodide (PI)/ RNase solution, and incubated for 30 minutes in the dark at room temperature in anticipation of flow cytometry [23 (link)]. PI stains total DNA and FITC-dUTP stains apoptotic cells.

Agarwal A., Mulgund A., Alshahrani S., Assidi M., Abuzenadah A.M., Sharma R, & Sabanegh E. (2014). Reactive oxygen species and sperm DNA damage in infertile men presenting with low level leukocytospermia. Reproductive Biology and Endocrinology : RB&E, 12, 126.

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TUNEL Assay for Tumor Apoptosis

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TdT mediated dUTP Nick End Labeling (TUNEL) assay for tumor sections were performed by using APO-DIRECT (BD Biosciences, CA, USA) following the manufacturer’s protocol. Briefly, tumor sections were treated with proteinase K (10 mg/ml) for 30 min. Next, tumor sections were washed three times with PBS and stained with the TUNEL reaction mixture overnight in cold box and washed three times with PBS. The slides were mounted with DAPI containing mounting medium. The slides were visualized by confocal microscopy and TUNEL positive cell were quantify by Image J software.

Mohammad N., Vikram Singh S., Malvi P., Chaube B., Athavale D., Vanuopadath M., Nair S.S., Nair B, & Bhat M.K. (2015). Strategy to enhance efficacy of doxorubicin in solid tumor cells by methyl-β-cyclodextrin: Involvement of p53 and Fas receptor ligand complex. Scientific Reports, 5, 11853.

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Comprehensive Protein Expression Analysis

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Antibodies against cdk6, cdk2, cdk4, cyclin B1, cyclin D1, cyclin D2, cyclin E2, p27, p21, PARP, Bcl-2, Bax, Bcl-XL, p-AMPK, AMPK, p-ACC, ACC, p-mTOR, mTOR, p-S6, S6, p62, LC3AB and caspase-3 were purchased from Cell Signaling Technology (Beverly, MA). Anti-mouse and anti-rabbit secondary antibody horseradish per-oxidase conjugate was obtained from Amersham Pharmacia Life Sciences. The Bio-Rad DC Protein Assay Kit was purchased from Bio-Rad, CA. Novex precast Tris-Glycine gels were obtained from Invitrogen. The APO-DIRECT™ was purchased from BD biosciences. AMPK kinase activity was assay was performed by AMPK kinase activity kit which was purchased from CycLex Co., Ltd. (Cat#CY-1182).

Akhtar N., Syed D.N., Khan M.I., Adhami V.M., Mirza B, & Mukhtar H. (2015). The pentacyclic triterpenoid, plectranthoic acid, a novel activator of AMPK induces apoptotic death in prostate cancer cells. Oncotarget, 7(4), 3819-3831.

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Apoptosis Analysis in Tumor Tissue

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Analysis of apoptotic cells in tumor tissue was performed by TdT-mediated dUTP Nick End Labeling (TUNEL) assay by using APO-DIRECT (BD Biosciences) following the manufacturer's protocol. Briefly, tumor sections were overlaid with mounting medium containing anti-fade (Santa Cruz Biotechnology). Further, these sections were subjected to confocal microscopy (Carl Zeiss, Heidelberg, Germany).

Singh S.V., Ajay A.K., Mohammad N., Malvi P., Chaube B., Meena A.S, & Bhat M.K. (2015). Proteasomal inhibition sensitizes cervical cancer cells to mitomycin C-induced bystander effect: the role of tumor microenvironment. Cell Death & Disease, 6(10), e1934-.

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TUNEL Assay for Apoptosis Detection

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Fine sections (4 μm) were prepared from formalin fixed paraffin embedded tumor tissue and fixed on glass slides. Slides were deparaffinized by xylene solution twice for 10 min and subsequently dehydrated in graded alcohol (100%, 95%, 70% and 50%) followed by staining with TdT mediated dUTP Nick End Labeling (TUNEL) assay for tumor sections were performed by using APO-DIRECT (BD Biosciences, CA, USA) following the manufacturer’s protocol. Briefly, tumor sections were washed three times with PBS and stained with the TUNEL reaction mixture overnight in cold box and washed three times with PBS. The slides were mounted with DAPI containing mounting medium and visualized by confocal microscopy.

Muhammad N., Ruiz F., Stanley J., Rashmi R., Cho K., Jayachandran K., Zahner M.C., Huang Y., Zhang J., Markovina S., Patti G.J, & Schwarz J.K. (2022). Mono- and diunsaturated fatty acids sensitize cervical cancer to radiation therapy. Cancer research, 82(24), 4515-4527.

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Sperm DNA Fragmentation Quantification

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Sperm DNA fragmentation was quantified using the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay kit (Apo-Direct; Pharmingen, San Diego, CA). After fixation with 3.7% paraformaldehyde for 30 minutes on ice, the spermatozoa were washed and resuspended in ice cold 70% ethanol, followed by another resuspension in phosphate-buffered saline. The specimens were centrifuged at 1600 rpm for 7 minutes, and the pellet was resuspended for 60 minutes at 37°C in 50 μL of solution containing terminal deoxynucleotidyl transferase enzyme, terminal deoxynucleotidyl transferase reaction buffer, fluorescein isothiocyanate tagged 20-deoxyuridine, 50-triphosphate nucleotides, and distilled water. After centrifugation, the cells were washed twice in a rinse buffer, resuspended in 0.5 mL of propidium iodide/RNase solution, and incubated for 30 minutes in the dark at room temperature in anticipation of the flow cytometry.

Kadioglu T.C., Aliyev E, & Celtik M. (2014). Microscopic Varicocelectomy Significantly Decreases the Sperm DNA Fragmentation Index in Patients with Infertility. BioMed Research International, 2014, 695713.

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