In Situ Cell Death Detection Kit, TMR red

Protocol tips

Upstream tips	Protocol tips	Downstream 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.	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

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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Manufacturer protocol

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

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