In Situ Cell Death Detection Kit, Fluorescein

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Fix cells in n 4% (w/v) paraformaldehyde for 1 h at room temperature Permeabilize cells with 0.5% (v/v) Triton X-100 in PBS for 1 h at 37 °C. Add Tunel mixture and incubate for 1 h at 37 °C in dark.

Protocol tips
Fix cells in n 4% (w/v) paraformaldehyde for 1 h at room temperature Permeabilize cells with 0.5% (v/v) Triton X-100 in PBS for 1 h at 37 °C. Add Tunel mixture and incubate for 1 h at 37 °C in dark.

Publication protocol

TUNEL assay was used to measure cell apoptosis. Cells were seeded on glass coverslips in six-well dishes, and at 48 h after differentiation, staining of fragmented DNA from apoptotic cells was carried out using an in situ Cell Death Detection Kit Fluorescein (Roche). Briefly, cells were rinsed with PBS followed by fixation in 4% (w/v) paraformaldehyde for 1 h at room temperature. Fixed cells were permeabilized by incubation with 0.5% (v/v) Triton X-100 in PBS for 1 h at 37 °C. The cells were rinsed again with PBS and incubated with 50 μl TUNEL reaction mixture per sample for 1 h at 37 °C. Following washes with PBS, samples were mounted on glass slides with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) and examined by inverted fluorescence confocal microscopy.

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