Annexin 5 fitc apoptosis detection kit

Cells were scored using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and an annexin V/FITC apoptosis detection kit (#AD10-10; Dojindo Laboratories), with 10,000 events acquired and analyzed using Cell Quest Software (BD Biosciences). iPSCs were cultured on plates that were uncoated or precoated with Matrigel or agarose for 48 h, followed by detachment of the iPSCs using trypsin to prepare a single-cell suspension and transfer of 2 × 10⁵ cells to a flow tube for washing with 2 mL PBS. Cells were resuspended in 200 µL of 1 × Annexin-V binding buffer to prepare a suspension at 1 × 10⁶ cells/mL. We then added 5 μL of Annexin-V/FITC solution and 10 μL propidium iodide-staining solution to the suspension, which was incubated in the dark for 15 min. Cells were analyzed within 1 h by flow cytometry. The experiment was repeated three times. Data were analyzed using FlowJo software (v.10.0; FlowJo LLC, Ashland, OR, USA). Annexin V-FITC⁻/PI⁺(Q1), annexin V-FITC⁺/PI⁺(Q2), annexin V-FITC⁺/PI⁻(Q3) and annexin V-FITC⁻/PI⁻(Q4) were necrotic, late apoptotic, early apoptotic and living cells, respectively. Their percent rates out of total cells (Q1 + Q2 + Q3 + Q4) were calculated, respectively.

An Annexin V-FITC Apoptosis Detection Kit (DOJINDO, Shanghai, China) was used for detecting apoptosis according to the instructions of the manufacturer. After cultured with recombinant TNF-α (10 ng ml⁻¹) and FasL (10 ng ml⁻¹) for 24 h or cultured for 6 days in the deprivation of any extra cytokines, the CAR-T cells were labeled by anti-human APC-CD56 (Clone TULY56, 17-0566-42), Annexin V, and propidium iodide, and then detected by a flow cytometer BD FACS Aria.

Podocyte apoptosis was also determined by the Annexin V-FITC Apoptosis Detection Kit (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. In brief, podocytes were washed with PBS, trypsinized, and re-suspended in binding solution, and then 5 μL of AnnexinV-FITC and 5 μL PI were added to 200 μL of the podocyte-containing solution and incubated at room temperature in the dark for 15 min. Apoptosis rates, representing both early apoptotic (Annexin V+/PI−) and late apoptotic (Annexin V+/PI+) podocytes, were detected with flow cytometry.

Apoptosis in ovarian cancer cells were analyzed with Annexin V-FITC Apoptosis Detection Kit (Dojindo Molecular Technologies) according to manufacturer's instructions. Briefly, cultured cells were trypsinized with 0.25% trypsin without EDTA, and stained with Annexin V FITC and PI solution. Stained cells were then subjected to flow cytometry analysis on a BD Accuri™ C6 (BD Biosciences, NJ).

Cells (3 × 10⁵ cells/well) were seeded in a 6-well plate overnight for attachment. siRNAs or plasmids were added to the cells for 48 h, followed by incubating in serum-free medium for another 24 h. To determine the apoptotic cell ratio, the cells were stained using an annexin V-FITC apoptosis detection kit (Dojindo, Kumamoto, Japan) and examined by flow cytometry for analysis. Data were analysed using FlowJo version 10.0.

The Cell Counting Kit-8 (CCK-8, DOJINDO, Shanghai, China) was used for cell proliferation experiments. Cells were inoculated on a 96-well plate at 2x10⁴ cells per well. Each group of cells was separately transfected with different oligonucleotides or plasmids using Lipofectamine™3000 and each group had 6 biological replicates. Next, MG-HS (7µL, 10¹⁰ CCU/mL) was utilized to infect DF-1 cells for 2 hours. At 12h, 24h, and 36h post-transfection, the cell proliferation curve was measured by the CCK-8 kit according to the manufacturer’s instructions. Transfection treatments were described above. Annexin V, FITC apoptosis detection kit (DOJINDO) was used to test the cell apoptosis. Each group was repeated three times.

Cell apoptosis and necrosis was quantitatively measured using Annexin V-FITC apoptosis detection kit (Dojindo, Kumamoto, Japan) by flow cytometry (FCM). Cells were harvested and washed by PBS, then dyed by FITC and PI (8μl/ml) for 30 min in room temperature before detected by FCM. Double-negative FITC-/PI-, single positive PI+, single positive FITC+ and double-positive FITC+/PI+ represented the living cells, mechanical injury cells, early phase apoptotic cells and late phase apoptotic or necrotic cells, respectively.