Annexin 5 fluos apoptosis detection kit

Annexin stain assay was conducted as previously described [19 (link)], with slight modification. Phosphatidylserine (PS) translocation was determined using annexin-V-Fluos apoptosis detection kit (Roche). To each 100 μl A549 cell suspension, 100 μl of Annexin staining buffer and 100 μl of Annexin-V-Fluos labelling solution (annexin-V: propidium iodide: annexin staining buffer (1:1:50 vol/vol/vol)) were added in 1.5 ml tubes. The BD Accuri flow cytometer was used to capture data from stained cells. Cells were analysed using the Accuri C6 gating and CFlow Plus v1.0 analysis software. For each sample 50,000 events were analysed in triplicate. The results are presented as percentage of apoptosis where cells positive for Annexin-V (FITC) in the lower right quadrant (early apoptosis) and upper right quadrant (late apoptosis) were gated.

One hundred thousand HL-60, KG-1 or primary AML cells treated with DNR, Ara-C, Flu and Eto as described above or un-treated were stained with human-specific primary monoclonal antibodies (mAbs) for CRT (AB92516; Abcam, Cambridge, UK), HSP70 (AB181606; Abcam) and HSP90 (AB13495; Abcam) at dilution ratios of 1:100, 1:230 and 1:250, respectively, in blocking solution (PBS/FBS 2%). After 30 min of incubation, the cells were washed with cold PBS and stained for another 30 min in the dark with secondary mAb Donkey Anti-Rabbit IgG-AlexaFluor 647 (AB150075, Abcam) diluted at 1:5000. After a final wash with cold PBS, the cells were stained with Ann-V by Annexin-V-FLUOS Apoptosis Detection Kit (Roche) for 15 min and then analyzed on flow cytometer BDAccuriC6 (BD Biosciences, Franklin Lakes, WI, USA). At least 10,000 events were analyzed. The cells stained only with secondary mAb for each condition were used as negative fluorescence control.

HL-60, KG-1 and primary AML cells were treated with DNR 500 ng/mL (Sigma-Aldrich, St. Louis, MO, USA), Ara-C 20 µg/mL (Sigma-Aldrich), Flu 70 µg/mL (Sigma-Aldrich) or Eto 20 µg/mL (Sigma-Aldrich) for 4 h, washed and used for DC pulsing or reseeded for another 20 h and then tested for apoptosis by Annexin-V-FLUOS Apoptosis Detection Kit (Roche, Basel, Switzerland), according to the manufacturer’s instructions, and flow cytometry staining (see below). ATP release and immunofluorescence staining (see Section 4.4 and Section 4.5, respectively) were performed only with HL-60 cell lines.

The Chinese Academy of Sciences’ Institute of Biochemistry and Cell Biology supplied the ovarian cancer cell line that was purchased. WZ10 was purchased from Sigma (St. Louis, MO, USA). Phospho-YAP (Ser127) (D9W2I) Rabbit mAb (#13008), MST1 (D8B9Q) Rabbit mAb (#14946), MST2 Antibody (#3952), LATS1 (C66B5) Rabbit mAb (#3477), Phospho-MOB1 (Thr12) (D2E3) Rabbit (mAb #8843), β-Actin (8H10D10) Mouse mAb (#3700), Cyclin D1 (E3P5S) XP^® Rabbit mAb (#55506), Oct-4A (C52G3) Rabbit mAb (#2890) and Vimentin (D21H3) XP^® Rabbit mAb (BSA and Azide Free) (#46173) were purchased from Cell Signaling Technology (Boston, MA, USA). The immunohistochemistry kit was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., LTD. Fetal bovine serum, antibiotics, DMEM medium (Gibco) and 5-Bromo-2-deoxyUridine (BrdU) cell proliferation assay kit were purchased from LifeTechnologies (Gaithersburg, MD, USA). YAP shRNA and the transfection kit Effectene^R Transfection Reagent were purchased from QIAGEN, USA. Annexin V-FLUOS Apoptosis Detection Kit was purchased from Roche (Basel, Switzerland). FACScalibur flow cytometer was purchased from Becton-Dickinson (Franklin Lake, NJ, USA). HybridMulti-Mode Microplate Readers Microplate Reader was purchased from Omega Biotek (Norcross, GA, USA).

The total number of cells and cell viability were determined by the trypan blue exclusion test (Sigma). Apoptotic cells in the populations were measured with a FACScan flow cytometer (Becton-Dickinson, San Jose, CA, USA) with the Annexin V FLUOS Apoptosis detection kit (Roche Molecular Biochemicals, Mannheim, Germany) according to manufacturer’s instruction.