Annexin 5 fitc detection kit

DAOY cells were seeded onto eight wells coverslips for 24 h in media. Rhodamine-GuaDex was prepared as previously [21 (link)]. The cells were PBS washed, incubated with Rhodamine-GuaDex at 5 µM for 1 h in PBS, and then washed twice with PBS. Cells were then incubated with 10 µL Annexin V-FITC detection kit (Sigma-Aldrich, St. Louis MO, USA) in PBS for 30 min at 4 °C dark and RT. After staining, the cells were washed carefully and prepared with DAPI mounting medium. A confocal microscope (Leica SP5) was used for the image analysis.

The apoptotic rate of L929 and RAW 264.7 cells was determined using an Annexin V-FITC detection kit (Sigma-Aldrich) according to the manufacturer’s protocol. At indicated time points, control and ECTV-infected L929 and RAW 264.7 cells grown in 12-well culture plates were collected, washed twice with cold PBS, and resuspended in 1× binding buffer. In the next step, 1 × 10⁵ cells contained in 100 μL of binding buffer were labeled with 5 μL Annexin V-FITC and 5 μL propidium iodide (PI) and incubated for 15 min at RT in the dark. Then, cells were resuspended in 400 μL of 1× binding buffer and analyzed by flow cytometry (λ_ex = 488 nm).
The kit includes Annexin V conjugated with FITC to label phosphatidylserine sites on the membrane surface, and PI to label the cellular DNA in necrotic cells where the cell membrane has been totally compromised. It can differentiate among early apoptotic cells (annexin V positive, PI negative), late apoptotic or necrotic cells (annexin V positive, PI positive), and viable cells (annexin V negative, PI negative).

C. neoformans cells were treated with the bioactive peptides (10d or 10o) at MIC value and incubated at 30 °C at 200× g for 12 h. The treated cells along with a batch of untreated cells were centrifuged at 2000× g for 10 min at 4 °C and suspended in cold phosphate-buffered saline (PBS), followed by 2–3 washings with PBS. The harvested treated and untreated cells were then incubated with zymolyase for 1 h at ambient temperature to obtain cell protoplasm. The staining of obtained protoplasm was done using an Annexin V-FITC detection kit (Sigma-Aldrich, Saint Louis, DE, USA). The protoplasm of untreated cells was divided into 4 tubes (2 mL) and into 3 tubes a different staining agent was added while one was kept unstained. Hence, four samples of untreated cells were prepared with (i) unstained cells alone, (ii) cells with propidium iodide (PI), (iii) cells with annexin V-FITC and (iv) cells with both PI and annexin V-FITC. Furthermore, two more samples were prepared where protoplasm of 10d- and 10o-treated cells were stained with both PI and annexin V-FITC. All 6 samples were then analyzed by using an FACS Calibur (Becton-Dickinson, San Jose, CA, USA) at excitation of 488 nm (for FITC) and 560 nm (for PI) band filter. Approximately 10,000 cells were used for counting [29 (link)].