Vybrant apoptosis assay kit 4

Oral cancer (CAL-27) cells (5 × 10⁵) were seeded in a 6-well plate and incubated for 24 h in presence of 5% CO₂. After incubation compound 1 was added at IC₅₀ and IC₇₀ concentration and further incubated for 48 h. Following incubation cells were harvested by using trypsin (0.05%) and pallet out after washing three times at 400×g for 5 min to remove media and trypsin. Cell pallets were suspended in 1 mL PBS and stained with Vybrant Apoptosis Assay kit # 4 (Invitrogen, USA) containing YO-PRO-1 and Propidium Iodide (PI) dye according to manufacturer protocol. Briefly, cells were incubated with 1 μL YO-PRO-1 stock solution (Component A) and 1 μL PI stock solution (Component B) for 30 min on ice. Later, cells were analyzed on FACSCalibur (Becton–Dickinson, USA). YO-PRO-1 and PI were excited at 488 nm, and fluorescence was measured at 530 and 620 nm, respectively. A total of 10,000 events were acquired from each sample. The percentages of live, apoptotic and dead cells were determined using CellquestPro software.

The percentages of red deer spermatozoa with viable plasma membranes and apoptotic-like changes in plasma membranes were determined with the Vybrant Apoptosis Assay Kit #4 (Molecular Probes Inc., Eugene, OR, USA) according to a previously described method [22 (link)] with some modifications. First, to visualize all cells, 1 µL of JC-1 was added to 200 µL of the sperm suspension (10 × 10⁶ sperm/mL) and incubated for 10 min at 37 °C. Then, 2 μL of YO-PRO-1 solution (100 μM) and 2 μL of PI (2 μM) were added to the stained sample and incubated for 5 min at 37 °C. After incubation, stained sperm cells were examined under a fluorescence microscope (Olympus, model BX41, Tokyo, Japan) at 600× magnification. A minimum of 200 cells per slide were examined in each aliquot. Four populations of spermatozoa were identified: with an unstained head, but with a visible mid-piece (viable sperm without apoptotic-like changes; YO-PRO-1⁻/PI⁻); with a green head (sperm with apoptotic-like changes in the plasma membrane; YO-PRO-1⁺/PI⁻), with a red and green head (moribund/dying sperm cells with dual fluorescence; YO-PRO-1⁺/PI⁺), and sperm with a red head (dead/necrotic cells; YO-PRO-1⁻/PI⁺).

The percentages of viable and plasma membrane apoptotic-like changes in spermatozoa were assessed using the Vybrant Apoptosis Assay Kit #4 (Molecular Probes, Inc., Eugene, USA), according to a previously described method [37 (link)]. Following incubation of sperm suspension (4 × 10⁶ spermatozoa/mL) in YO-PRO-1 (100 µM) and PI (2 μM) solutions for 15 min at 37 °C, aliquots of the stained sperm cells were examined at 600× magnification under a fluorescence microscope (Olympus CH 30). A minimum of 100 cells per slide were examined in each aliquot, and three sub-populations were identified: viable spermatozoa categorized as negative for both YO-PRO-1 and PI (YO-PRO-1⁻/PI⁻), plasma membrane apoptotic-like changes in spermatozoa (moribund spermatozoa) were categorized as positive for YO-PRO-1⁺ but negative for PI⁻ (YO-PRO-1⁺/PI⁻), and dead spermatozoa, which were positive for both YO-PRO-1 and PI (YO-PRO-1⁺/PI⁺).