7 aminoactinomycin d 7 aad

Flow cytometry studies were performed on FACSCalibur or FACSAriaII (BD Biosciences) using CellQuest v5.2.1 or FlowJo v10.0 analysis software to monitor the following:
Fas receptor. HeLa cells were reacted with APC-conjugated mouse anti-human CD95 (DX2, BD Biosciences); mouse anti-human IgG-APC was used as an isotype control. The geometric mean of fluorescence intensity (MFI) was determined for each condition.
Apoptosis. HeLa cells were treated overnight with 100 ng anti-Fas activating antibody (CH-11, Millipore), washed with PBS, incubated with 7-amino-actinomycin D (7-AAD, Millipore) for five minutes and the percent 7-AAD positive cells determined.

Standard or TIC-enriched PANC1 cultures were treated with CuO-NPs (0–10 µg/ml Cu) for 24 hours. For the analysis of apoptosis, cells were detached and subsequently immunostained with anti-prominin-1 (CD133, 1:250, Macs MiltenyiBiotec, Germany) and Annexin V-FITC antibody in the proper binding buffer (MBL, Japan), as recommended by the manufacturer. Next, 7-Aminoactinomycin D (7-AAD, Sigma-Aldrich, USA) was added to the samples at a final concentration of 1 µg/ml. For the analysis of cell cycle, cells were fixed with 70% ethanol, and subsequently stained with 40 μg/ml propidium iodide (PI, BioLegend) in the presence of 10 μg/ml RNase (Sigma Aldrich, Israel). Cells were acquired by CyanADP flow cytometer (Beckman Coulter, Nyon, Switzerland) and data was analyzed by FlowJo software7.6. (San Diego, CA).

Spleen cells were incubated with anti-mouse CD16/CD32 mAb (2.4G2; BD Biosciences, San Jose, CA) in FACS buffer (HBSS containing 2% FCS) for FcR blocking for 15 min on ice, and then incubated with BV421-conjugated anti-mouse CD3 mAb (145-2C11; Biolegend, San Diego, CA), APC-conjugated anti-mouse CD8 mAb (53-6.7; Biolegend), PE-conjugated anti-mouse CD4 (GK1.5 Biolegend) or Siglec F (E50-2440; BD Biosciences) mAb, PE-Cy7-conjugated anti-mouse CD19 mAb (6D5; Biolegend) or Gr1 (RB6-8C5; Affymetrix eBioscience, San Diego, CA) mAb for 20 min on ice. The expression profile of each cell surface marker on 7-aminoactinomycin D (7-AAD; Sigma-Aldrich Co. LLC, St. Louis, MO) -negative cells was analyzed on a MACSQuant Analyser (Miltenyi Biotec, Bergisch Gladbach, Germany) with MACSQuantify Software (Miltenyi Biotec) and FlowJo software (Tree Star Inc., Ashland, OR).

Bone marrow cells from untreated animals were collected as described above. First, we measured the expression of FPR2 in LSK cells. The anti-FPR2-FITC antibody was used (Bioss, Massachusetts, USA) to quantify this expression by flow cytometry. Afterwards, bone marrow cells were treated with rAnxA1 (100 nM) for 12 h, and viability of the LSK population determined using 7-aminoactinomycinD (7-AAD; Sigma Aldrich, USA); cell cycle phases and expression of Ki67 and Notch-1 were also evaluated. These quantifications were carried out by flow cytometry. Cells were fixed in 2% paraformaldehyde for 30 min, washed with 0.1 M glycine, and permeabilized with 0.001% Triton X-100. Subsequently, 2 × 10⁶ cells were incubated with primary anti-cyclin B1, anti-Notch-1 or anti-Ki67 (Cell Signaling Technology) antibodies for 2 h, and then 40 min with secondary rabbit Anti-IgG Alexa Fluor 488 (Molecular Probes/Invitrogen, USA). Analyses were performed on the LSK population gated as described above, using an Accuri C6 flow cytometer (Becton Dickinson, USA) and the FlowJo software.

Cells were collected by trypsinization after treated with silver ion medium by 5hr, 21hr, and 48hr. Immediately, cells were stained with 7-aminoactinomycin D (7-AAD; Sigma Chemical Co., St. Louis, MO) and 20nM of SYTO 13 (Molecular Probes, Eugene, OR) in PBS for 30 minutes in the dark. 7-AAD is a cell impermeable DNA binding dye that excites at 488 nm and emits in the far red, with an emission peak at approx 670 nm. The cell-permeable SYTO 13 is a green fluorescent nucleic acid dye that excites at 488 nm and emits at 509 nm. Flow cytometry was performed with an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA) that can detect SYTO 13 staining by FL1 (533/30 nM) and 7-AAD by FL3 (>670 nm), registered on a log scale. A threshold of channel 400,000 on FSC-H was set to gate out light scatter and fluorescence signals caused by debris in samples. The experiment was repeated four times with non-stained control. Samples were analyzed as: live normal cells–positive stain for SYTO 13; Necrotic cells–positive stain for SYTO 13 and 7-AAD.

EB were collected as previously described, the resulting single-cell suspension was incubated with the fluorochrome-conjugated antibodies (mAbs): CD34-APC, CD43-FITC and CD45-PE (antibodies from BD Biosciences).
Cells were incubated with selected antibodies for 30 minutes on ice, washed and resuspended in PBS containing 5% FCS and 7-aminoactinomycin D (7AAD; 1μg/mL) (Sigma Aldrich) to exclude dead cells. Isotype matched FITC-, PE- and APC-conjugated irrelevant mAbs (BD Biosciences and Beckman Coulter). Analysis was carried out with a FACSCalibur flow cytometer using CellQuest Pro Analysis software (Becton-Dickinson).

Cells were harvested, washed once with cold phosphate-buffered saline (PBS), and stained with FITC- or Alexa Fluor 647-conjugated AxV (640906 and 640912; BioLegend) and PI (2.5 μg/mL; Molecular Probes) or 7-Aminoactinomycin D (7AAD) (5 μg/mL, A9400; Sigma-Aldrich). In apoptotic cells, the membrane phospholipid phosphatidylserine translocates from the inner to the outer side of the plasma membrane, to which flurochrome-conjugated AxV with its high affinity to phospholipids can bind and detect apoptosis. By the addition of PI, which can enter the cell on loss of membrane integrity, early and late apoptosis can be distinguished. After incubation for 15 min at room temperature, cells were analyzed by flow cytometry on FACSCanto™ II (BD Biosciences).