Human prostate carcinoma LNCaP and 22Rv1 cells were obtained from the American Type Culture Collection (Manassas, VA). LNCaP and 22Rv1 cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin G and 100 μg/ml streptomycin sulfate at 37°C in a humidified 5% CO2 incubator. Media and other cell culture materials were from Invitrogen Corporation (Gaithersburg, MD). Cells were treated with different concentrations of silibinin (5–200 μM in medium), dissolved originally in dimethyl sulfoxide (DMSO), for descried time periods. An equal amount of DMSO (vehicle) was present in each treatment, including control; DMSO concentration did not exceed 0.1% (v/v) in any treatment. Antibodies for HIF-1α, HIF-1β, PHD1, PHD2, FIH (factor inhibiting HIF), phosphorylated and total ACC, and FASN were from Cell Signaling (Beverly, MA). Ki-67 and CD31 antibodies were from Abcam (Cambridge, MA), cyclin D1 antibody was from Thermo Scientific (Fremont, CA), TUNEL kit was from Promega (Madison, WI), PSA (prostate specific antigen) antibody was from DAKO (Carpinteria, CA), and HIF-1α antibody used for immunohistochemistry (IHC) was from Santa Cruz Biotechnology (Dallas, TX). Enhanced chemiluminescence (ECL) detection system was from GE healthcare (Buckinghamshire, UK). All other reagents were obtained in their commercially available highest purity grade.
Tunel kit
Manufactured by Promega
Sourced in United States
The TUNEL kit is a laboratory product designed to detect DNA fragmentation, a hallmark of apoptosis or programmed cell death. It utilizes terminal deoxynucleotidyl transferase (TdT) to label the free 3'-OH ends of fragmented DNA, allowing for the visualization and quantification of apoptotic cells.
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Lab products found in correlation
Lsm 700 meta, by Zeiss (4 mentions)
Lsm image browser software, by Zeiss (3 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Hif 1α antibody, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence ecl detection system, by GE Healthcare (1 mentions)
Lncap, by American Type Culture Collection (1 mentions)
Dm4000b, by Leica (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Fluorescence microscopy, by Olympus (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Dmla microscope, by Leica (1 mentions)
Ix71 cts, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Promega (1 mentions)
Inverted fluorescence microscope, by Olympus (1 mentions)
H e kit, by Nanjing Jiancheng (1 mentions)
Ab125066, by Abcam (1 mentions)
Chromomycin a3, by Merck Group (1 mentions)
Chloroquine, by Merck Group (1 mentions)
56 protocols using tunel kit
1
Silibinin Inhibits Prostate Cancer Cells
2
Apoptosis Detection in Hepatic Cells
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Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay was used for in situ detection of apoptotic cells with a commercially available TUNEL kit (Promega, Madison, USA). Propidium iodide (PI) 1 μg/ml was added as a nuclear counter stain. The number of TUNEL-positive hepatic cells was counted and average TUNEL positive cells per cross sectional area were recorded.
3
Apoptosis Analysis in Organ Tissues
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Apoptosis in the livers, spleens, and kidneys was identified by a terminal-deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) kit (Promega Corporation, Fitchburg, WI, USA). Paraffin sections of kidney, spleen, and liver tissues were deparaffinized with xylene and rehydrated in a series of graded ethanol. Proteinase K (20 mg/mL; Sigma-Aldrich Co., St Louis, MO, USA) was used for cell membrane perforation at 37°C for 30 minutes. The tissue sections were then incubated with a reaction mixture of terminal deoxynucleotidyl transferase at 37°C for 1 hour. Finally, 4′,6-diamidino-2-phenylindole was added to stain the nucleus, and the apoptotic cells were detected by fluorescence microscopy (DM4000B, LEICA, Wetzlar, Germany).
4
Luteolin/MPEG-PCL Micelles Induce Apoptosis
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Apoptosis of U87 tumors induced by Luteolin/MPEG-PCL micelles was detected by TUNEL staining. When tumor tissues were sectioned, a TUNEL kit (Promega, Madison, WI, USA) was used to analyze apoptotic cells within tumors following the manufacturer's protocol. Five equal-sized tumor sections were detected. TUNEL positive cells were observed under a fluorescent microscope (×400).
5
TUNEL Apoptosis Detection Protocol
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Apoptosis was detected using a TUNEL kit (Promega, Fitchburg, WI, USA). Briefly, sections were deparaffinized and rehydrated, rinsed in 0.1 M PBS for 10 minutes, and then reacted with proteinase K (20 μg/mL) for 10 minutes at 25°C. After washing in PBS, the sections were incubated with equilibration buffer (200 mM potassium cacodylate, 25 mM Tris-HCl, 0.2 mM DTT, 0.25 mg/mL bovine serum albumin, 2.5 mM cobalt chloride) for 10 minutes at room temperature. The specimens were then incubated in labeling reaction mix (terminal deoxynucleotidyl transferase and deoxynucleotides) for 1 hour at room temperature in the dark. The sections were then incubated with 2× saline citrate (300 mM NaCl, 30 mM sodium citrate) for 10 minutes at 25°C, washed in PBS, and reacted with 4′,6-diamidino-2-phenylindole for 15 minutes at 25°C in the dark. Fluorescence microscopy (Olympus) was used to acquire images. The positive control was produced by adding DNaseI for 5 minutes at 25°C. PBS instead of terminal deoxynucleotidyl transferase was used for the negative control.
6
Apoptosis Detection via TUNEL Assay
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Internucleosomal DNA fragmentation was detected using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) kit (Promega, Madison, WI, USA). In short, cells were cultured on 18 mm coverslips. After the indicated treatment, the fixed cells were incubated with TUNEL reaction mixture for 1 h at 37 °C according to the manufacturer’s protocol and then viewed under a fluorescence microscope (ZEISS, Jena, Germany). TUNEL-positive nuclei were counted in five randomly chosen fields per coverslip three times; then, the apoptotic percentage was calculated by comparing the TUNEL-positive counts with the total cell nuclei, determined by Hoechst 33342 counterstaining.
7
Immunohistochemistry and TUNEL Staining Protocol
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Antigen retrieval was performed on tissue sections using a sodium citrate based buffer (Vector Laboratories, Burlingame, CA). Tissues were blocked with 5 % BSA/0.5 % Tween and incubated with primary antibodies overnight. For immunohistochemistry with MMP-1 (MAB901; R&D Systems) and EREG (AF1195; R&D Systems) and biotinylated anti-mouse or anti-goat secondary antibody (Vector Laboratories) were used following heat based antigen retrieval (Vector Laboratories). Antibody was detected using a Vectastain ABC kit (Vector Laboratories), visualized with DAB (Vector Laboratories) and counterstaining was performed with hematoxylin. For immunofluorescence, sections were stained using the TUNEL kit (Promega, Madison, WI) according to the manufacturer’s protocol.
8
Laser-Induced Apoptosis in Nude Mice
9
Curcumin Induces Apoptosis in A375 Tumors
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Apoptosis of A375 tumors induced by Curcumin/MPEG-PLA micelles was detected by TUNEL staining. When tumor tissues were sectioned, a TUNEL kit (Promega, Madison, WI, USA) was used to analyze apoptotic cells within tumors following the manufacturer’s protocol. Five equal-sized tumor sections were detected. TUNEL positive cells were observed under a fluorescent microscope (×400).
10
TUNEL Assay for Apoptosis Detection
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A DeadEnd Fluorometric transferase dUTP nick end labeling (TUNEL) kit and 4’-6-diamidino-2-phenylindole (DAPI) and were obtained from Promega (Madison, WI, USA). DAPI and TUNEL assays were performed according to the instructions of the manufacturer. A375 and A2058 cells (1 × 105 cells/well, 12-well microplate) were plated in each well of the plate and treated with different concentrations of (+)-bornyl p-coumarate (0, 12 and 24 µM). The (+)-bornyl p-coumarate-treated cells and untreated cells were fixed with 4% paraformaldehyde (dissolved in PBS) before staining. Cells after DAPI and TUNEL staining were then photographed under a fluorescence microscope (Olympus IX71 CTS).
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