In Situ Cell Death Detection Kit, TMR red

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Fix cell samples with Fixation solution for 1 h. Incubate in Permeabilisation solution (0.1% Triton X-100 in PBS) for 2 min on ice. Label and incubate for 60 min at +37°C in a humidified atmosphere in the dark	If low labelling is found, reduce fixation time or try 2% buffered paraformaldehyde

Protocol tips
Fix cell samples with Fixation solution for 1 h. Incubate in Permeabilisation solution (0.1% Triton X-100 in PBS) for 2 min on ice. Label and incubate for 60 min at +37°C in a humidified atmosphere in the dark
Downstream tips
If low labelling is found, reduce fixation time or try 2% buffered paraformaldehyde

Publication protocol

After C2C12 cells were subjected to LMS or HMS for 12 and 24 h, they were washed with phosphate-buffered saline (PBS). Then, the terminal deoxynucleotidyl TUNEL assay was performed for adherent cells with the In Situ Cell Death Detection Kit, TMR red (Roche), according to the manufacturer’s instructions. After the TUNEL reaction, nuclei were counterstained with DAPI according to the manufacturer’s instructions. Samples were analyzed under a fluorescence microscope, Nikon ECLIPSE E400, using a 520–550 or 330–385 nm excitation filter, and a 580 or 420 nm band pass filter for TUNEL and DAPI assays, respectively. Images of four microscopic fields at 100-fold magnification were captured randomly, and the TUNEL-positive cells and DAPI-positive nuclei were counted manually to calculate the percentage of apoptotic cells to total cells. Data are presented as the percentage of three 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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