In Situ Cell Death Detection Kit, TMR red

TUNEL assay cell type - Rabbit synovial fibroblasts

Experiment
TUNEL assay cell type - Rabbit synovial fibroblasts
Product
In Situ Cell Death Detection Kit, TMR red from Sigma-Aldrich
Manufacturer
Sigma-Aldrich

Protocol tips

Protocol tips
Fix cells in fixation solution for 1 h at +15 to +25°C.

Add tunel reaction mxture and incubate for 60 min at +37°C in a humidified atmosphere in the dark.

Downstream tips
If Nonspecific labeling is noticed, try formalin or glutaraldehyde; reduce concentration of TdT by diluting it 1:2 up to 1:10 with TUNEL Dilution Buffer.

If high background, reduce concentration of labeling mix
to 50% by diluting with TUNEL Dilution Buffer

If Low labeling is noticed, increase incubation time. or incubate at higher temperature
(e.g., +15 to +25°C).

Analyse using a 520–550 nm or 330–385 nm excitation filter, and a 580~ nm or 420~ nm

Publication protocol

After HIG-82 cells were incubated in a 24-well plate with Ham’s F12 medium containing vehicle buffer or 100 μg/ml β2-m fibrils or r-β2-m monomer for 2 days, they were washed with PBS (-) two times. Then, the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay was performed for adherent cells with In Situ Cell Death Detection Kit, TMR red (Roche) according to the manufacturer’s instructions. As a positive control, cells were incubated with 1 000 unit/ml DNase I recombinant, RNase-free (Roche) for 10 min at room temperature to break DNA strand, prior to labeling procedure. After TUNEL reaction, nuclei were counterstained with DAPI (DAPI solution, PromoCell, Heidelberg, Germany or DAPI Nucleic Acid stain, Lonza Walkersville, Inc., Walkersville, MD, USA) according to the manufacturer’s instructions. Samples were analyzed under a fluorescence microscope (IX70, Olympus) equipped with a CCD camera (DP70, Olympus) using a 520–550 nm or 330–385 nm excitation filter, and a 580~ nm or 420~ nm band pass filter for TUNEL and DAPI assays, respectively. Images of 4 microscopic fields at 100-fold magnification were captured randomly, and the number of TUNEL-positive cells and DAPI-positive nuclei was counted manually to calculate the percentage of apoptotic cells to total cells. Data were presented as a dot plot of the percentage of five independent experiments with the mean value.

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Discussion

Discussion

5 years ago

Author: Ercole Udinesi Italy

Are 10 week old samples in formalin still usable?

I have left my tumor samples in formalin for an extended period of time (around 10 weeks). Do you think I will be able to use them for TUNEL assay and get results? Thank you for your help in advance

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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing TUNEL assay cell type - Rabbit synovial fibroblasts using In Situ Cell Death Detection Kit, TMR red from Sigma-Aldrich.

Paper title
Endocytosed β2-Microglobulin Amyloid Fibrils Induce Necrosis and Apoptosis of Rabbit Synovial Fibroblasts by Disrupting Endosomal/Lysosomal Membranes: A Novel Mechanism on the Cytotoxicity of Amyloid Fibrils
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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for In Situ Cell Death Detection Kit, TMR red below.

Download PDF Download manufacturer protocol

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