Alexa fluor 488 annexin 5 dead cell apoptosis kit with alexa fluor 488 annexin 5 and pi for flow cytometry

5 × 10⁴ KGN or Cov434 cells were seeded in 10 cm culture dish for up to 6 days. Proliferation rate was measured using cell-counting kit-8 (DOJINDO) according to the manufacturer’s protocol. Annexin V/propidium iodide (PI) staining was performed for the detection of apoptotic cells. After desired treatment, 1 × 10⁶ cells were collected and washed twice with ice-cold PBS. The cells were then stained using the Alexa Fluor^®488 annexin V/Dead Cell Apoptosis Kit with Alexa^® Fluor 488 annexin V and PI for Flow Cytometry (Invitrogen, CA, USA) according to the manufacturer’s guidelines. The untreated cells served as a negative control for the double staining. Transwell (Costar) was used for migration and invasion assays. Mytomycin C was used for cell synchronization for 24 h. 2 × 10⁴ KGN cells were seeded into the upper chamber with serum-free medium and serum-contained medium was added to the lower wells (Matrigel was coated in upper chamber for invasion assay). After 24 and 48 h, the upper chamber was washed with PBS twice and fixed by 4% paraformaldehyde for 20 min, and then 1% crystal violet was used to stain migrated and invasive cells.

NUGC4 and SGC7901 cells were incubated overnight in 6-well culture plates at a density of 1 × 10⁵ cells/well, and treated differently to analyze cell proliferation. The different groups were as follows: saline control, blank NPs, blank TNPs, free AMO-21, trastuzumab, AMO-21-Lipofectamine 2000 + trastuzumab, AMO-21-NPs + trastuzumab, AMO-21-TNPs for72 h. The cells were processed as described in the cell apoptosis kit (Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit with Alexa Fluor 488 annexin V and PI for Flow Cytometry, Invitrogen), followed by flow cytometry (BD Biosciences, Sparks, MD, USA).

Cells were seeded at 1.5 × 10⁵ cells/ml, and OGD was performed. The number of apoptotic cells was assessed using an annexin V-FITC/PI assay according to the manufacturer’s instructions (Alexa Fluor^® 488 annexin V/Dead Cell Apoptosis Kit with Alexa^® Fluor 488 annexin V and PI for Flow Cytometry, Invitrogen, Carlsbad, CA, USA). Briefly, following OGD injury, the cells were trypsinized without EDTA, harvested, and washed in cold PBS twice. Cells were then treated with annexin V/PI solution in 1 × annexin-binding buffer, and thereafter analyzed using a BD FACS Caliber flow cytometer (BD Biosciences, San Jose, CA). Three separate experiments were performed [26 (link)].

The apoptotic effect of C2-Cer on the HNSCC cells was determined via BD LSRII flow cytometry (BD Bioscience, Bedford, MA, USA). Cells (2 × 10⁵) were incubated as described above and suqsequently collected. Cell suspensions were treated according to the experimental protocols (Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit with AlexaFluor 488 annexin V and PI for Flow Cytometry, Invitrogen, Carlsbad, CA, USA) and observed under a Leica MM AF upright fluorescence microscope (Leica, Heidelberg, Germany). All experiments were repeated three times.

Cells were seeded in 60 mm culture plates (7.5 × 10⁵), infected with CTL or Staufen1-shRNA lentivirus and maintained at 70% confluency. Cells were co-stained 72 h post-secondary infection using the Alexa Fluor^® 488 Annexin V/Dead Cell Apoptosis Kit with Alexa Fluor^® 488 annexin V and PI for Flow Cytometry (Thermo Fisher Scientific, Ontario, Canada) according to manufacturer instructions. Stained cells were analyzed using the Beckman MoFlo^® Astrios^TM or the BD LSRFortessa^TM flow cytometers. Data analysis was performed using FlowJo software.

Cells were seeded in 60 mm culture plates (as stated above), infected with CTL or Staufen1-shRNA lentivirus and maintained at 70% confluency. Cells were co-stained 48 h post-secondary infection using the Alexa Fluor_® 488 Annexin V/Dead Cell Apoptosis Kit with Alexa Fluor_® 488 annexin V and PI for Flow Cytometry (Thermo Fisher Scientific, Ontario, Canada) according to manufacturer instructions. Stained cells were analyzed using the BD FACSCelesta™ or the BD LSRFortessa™ flow cytometers. Data analysis was performed using FlowJo software.