Mock and infected LC, SC, or STC grown on glass coverslips were fixed with 4% PFA, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 5% bovine serum albumin in PBS. Cells were then incubated with primary antibodies against anti-Spike (GeneTex, GTX632604 at 1:500 dilution), followed by fluorophore-conjugated secondary antibody (Invitrogen Alexa Fluor 488-conjugated sheep anti-rabbit, 1:5000 dilution), and examined using an Axiocam MR camera mounted on a Zeiss Axiovert 200 microscope. TUNEL assay was performed using the Promega Fluorometric TUNEL System according to the manufacturer’s instructions. Undifferentiated spermatogonia were also stained for the well-established cell-specific marker, ubiquitin carboxyl-terminal esterase L1 (UCHL1) [31 (link)] using rabbit anti-human UCHL1 (Sigma, HPA005993 at 1:1,000 dilution). The secondary antibody was Alexa Fluor 594-conjugated goat anti-rabbit (Invitrogen; 1:5000 dilution).
Fluorometric tunel system
Manufactured by Promega
Sourced in United States
The Fluorometric TUNEL System is a laboratory tool used to detect and quantify apoptosis, a type of programmed cell death, in biological samples. It utilizes fluorescent labeling to identify DNA fragmentation, a hallmark of apoptosis. The system provides a reliable and sensitive method for researchers to analyze and study cell death processes.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated sheep anti rabbit, by Zeiss (1 mentions)
Axiocam mr camera, by Zeiss (1 mentions)
Hpa005993, by Merck Group (1 mentions)
Gtx632604, by GeneTex (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Axiovert 200 microscope, by Zeiss (1 mentions)
Caspase 9, by Proteintech (1 mentions)
Hrp conjugated anti rabbit or anti mouse antibody, by Proteintech (1 mentions)
Caspase 3, by Proteintech (1 mentions)
Bcl 2, by Proteintech (1 mentions)
Annexin 5 fitc pi, by Keygen Biotech (1 mentions)
Eclipse ti e inverted microscope, by Nikon (1 mentions)
Hoechst stain solution, by Merck Group (1 mentions)
Eclipse te2000 u, by Nikon (1 mentions)
7 protocols using fluorometric tunel system
1
Immunostaining and TUNEL Assay for SARS-CoV-2
2
Immunohistochemical Detection of Autophagy and Apoptosis in Testis
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Briefly, after blocking with 0.5% BSA (Bovine serum albumin) in PBST (Phosphate Buffered Saline with Tween 20), paraffin-embedded testis slices were incubated with LC3 antibodies at 4°C overnight (Anti-LC3, MBL Inc, #PM036). Biotin-conjugated secondary antibodies (CST, #8114) and ABC-DAB staining kit (Vecta ABC and DAB standard kit, #PK-400 and SK-4105) were used for antibody localization and the nuclei were counter-stained with hematoxylin. For TUNEL staining, tissues were dewaxed and detected with Fluorometric TUNEL System (Promega) according to the working manual.
3
Apoptosis Evaluation in Cells
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DOX was purchased from Shenzhen Main Luck Pharmaceuticals Inc. (Shenzhen, China). Caspase-9, Caspase-3, Bcl-2, Bax antibodies and secondary antibodies directed against rabbit or goat (HRP-conjugated anti-rabbit or anti-mouse antibody) were purchased from Proteintech Group (Chicago, IL, USA). ROS detection kit for flow cytometry was purchased from Enzo R Life Sciences (Farmingdale, NY, USA). Fluorometric TUNEL system was purchased from Promega (Madison, WI, USA). All chemicals and solvents were of analytical grade.
4
Apoptosis Quantification by Flow Cytometry
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Apoptosis was assessed using the Fluorometric TUNEL System from Promega, as per the manufacturer’s instructions. Briefly, the indicated cells were treated and stained using the apoptosis detection kit Annexin V-FITC/PI from KeyGen (China) according to the manufacturer’s instructions. Finally, apoptosis was detected by flow cytometry. Analysis of more than 1 × 106 cells was performed per measurement.
5
Cardiac Apoptosis Quantification
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Cardiac apoptosis was assessed by fluorometric TUNEL system (Promega, Madison, WI, catalog number G3250). According to manufacture’s protocol, tissue sections or cells were fixed and labeled with TdT reaction mixture for 1 hr at 37°C. After the reactions were terminated, the slides were sealed with and then examined under Nikon Eclipse Ti-E inverted microscope at 20x magnification. Positive nucleus staining was obtained by propidium iodide (PI) in tissue sections and by DAPI in cultured cardiomyocytes.
6
Fluorometric TUNEL Assay for Apoptosis
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Fluorescence of apoptotic cells was detected via terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick-end labeling assay using the fluorometric TUNEL system (Promega, Madison, WI, USA). MG-63 and U2OS cells were plated on 6-well plates at a density of 3 × 105 per well and incubated for 24 h, respectively. Cells were fixed with 4% formaldehyde for 25 min and permeabilized using 0.5% Triton X-100 for 10 min. Cells were then treated with 50 μL TdT enzyme buffer. Cell nuclei were stained using 5 μL Hoechst Stain Solution (Sigma-Aldrich). Labeled strand breaks were visualized using a fluorescence microscope (Nikon Eclipse TE 2000-U, Tokyo, Japan)
7
Quantification of Apoptotic Cells by TUNEL
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TUNEL assay was performed by using the Fluorometric TUNEL System (Promega, USA) according to the protocol provided by the manufacturer. Briefly, 50 μL of rTdT Incubation Buffer was added to the fixed cells on slides. Then, the slides were incubated under humidified atmosphere for 60 min at 37°C in darkness and rinsed three times with 2 × SSC for 5 min each time. The slides were subsequently stained with DAPI and observed under a fluorescence microscope (BX51, Olympus, Japan). TUNEL-positive cells were indicated by the emission of green fluorescence and the nuclei were visualized by the blue fluorescence. Ratio of the number of TUNEL-positive neurons to the total number of nuclei was calculated.
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