Annexin 5 and 7 aad

First, we analyzed cell death by quantifying cell attachment with Countess II Automated Cell Counter (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, the solution covering the cells was removed and the cell suspension was washed once with PBS for further analysis. Attached cells were detached (0.25% trypsin) at 37 °C, washed once with PBS, and an aliquot (10 µL) of cell suspension was quantified using the cell counter. Subsequently, we analyzed cell apoptosis/necrosis using Annexin-V and 7-AAD (BD Biosciences, San Jose, CA, USA) for cell staining. Their fluorescence was detected using a BD FACSCanto II instrument, and the data were analyzed with BD FACSDiva version 8.0.1 (BD Biosciences, San Jose, CA, USA). We also analyzed cell death by detection of the release of lactate dehydrogenase (LDH) enzymes in cell culture media. Enzyme activity was measured using Dimension Vista Intelligent Lab System (Siemens Healthcare Diagnostics, Norwood, MA, USA), according to the manufacturer’s instructions.

The K562, MM.1S, and CD20⁺ 721.221 cell lines were obtained from ATCC and propagated in RPMI 1640 supplemented with 10% heat-inactivated FBS (Sigma-Aldrich) and 2 mM glutamine (Life Technologies). The following reagents were used: anti-CD56 (NCAM-1), anti-CD3 (UCHT1), anti-CD16 (3G8), anti-NKG2D (1D11), anti-TRAIL (RIK-2), anti-NKp30 (p30-15), anti-NKp46 (29A1.4), IgG1 (MOPC21), and Annexin V and 7-AAD from Becton Dickinson (BD); anti-KIR2DL1/DS1 (EB6), anti-KIR2DL2/3/DS2 (GL183), and anti-NKG2A (Z199) from Beckman Coulter; the anti-CD107a (H4A3), anti-KIR3DL1 (Dx9), anti-CD57 (HCD57), anti-2B4 (C1.7), anti-CD34 (581), anti-CCR7 (G043H7), IgG2a (MOPC-173), and BV650-streptavidin from Biolegend; the anti-Lir-1 (HP-F1) from eBioscience; the anti-NKG2C (134591) from R&D Systems; LIVE/DEAD viability marker from Life Technologies; biotinylated anti-KIR3DL2 (Dx31) from UCSF; rituximab (Rituxan) from Genentech; and off-the-shelf eGFP mRNA and custom-made CD34, CD16, and CCR7 mRNAs from TriLink Biotechnology.

Caski and SiHa cell lines were plated (1 × 10⁶/plate) and allowed to attach overnight. Next morning, cells were treated with different concentrations of ormeloxifene for 24 hours, both floaters and adherent cells were collected, washed twice with PBS and stained with Annexin V and 7AAD (BD Biosciences, CA, USA) 5 µL of each/100 µL of cell suspension for 20 mins in the dark at room temperature. After the incubation, cells were analyzed with Accuri C6 flow cytometer (BD Biosciences, CA, USA) in FL2 channel.

Cell line and primary patient or healthy mast cells were treated with SAHA (Cayman Chemical, Ann Arbor, Michigan), Panobinostat (Sigma Aldrich) or Romidepsin (Sigma Aldrich), all dissolved in DMSO, or Valproic acid (Sigma Aldrich) dissolved in distilled water. Control experiments were incubated with DMSO vehicle alone. Viability determination was done with either trypan blue exclusion or using flow cytometry Annexin V-FITC propidium iodide Apoptosis detection Kit according to the manufacturer’s protocol (eBioscience). For quantification of KIT expression the HMC-1.2 cells were incubated with 1% human CD117 (A36E2) (Bioledgend) while for the patient samples we used CD117 (A3C6E2)-PE (Miltenyi Biotec, Bergisch Gladbach, Germany) or 1% mouse IgG2b isotype control PE (Bioscience, San Jose, CA, USA) for 15 min at 4°C. Enriched CD117 high positive bone marrow cells have previously been described to be highly enriched for mast cells, thus we defined CD117 high positive cells as mast cells [36 (link)]. All samples were analyzed on a FACSCalibur flow cytometer (Becton Dickinson), employing the Cellquest software, and the data acquired analyzed with the FlowJo 7.6 program.
For ROSA cells, apoptosis staining was performed with Annexin V and 7-AAD (BD Bioscience) in Annexin V Binding Buffer (BD Bioscience).

Cells were stained with fluorophore-conjugated antibodies at 4°C for 10 min (or for 45 min for anti-CXCR4 and anti-CXCR7 antibodies), washed and re-suspended in 1X PBS containing 10% FBS. A FACSCalibur flow cytometer (Becton Dickinson) or a LSRFortessa flow cytometer (Becton Dickinson) were used to acquire data and FlowJo software (TreeStar) was used for data analysis. The following monoclonal antibodies were purchased from BD Biosciences: CD3 (SK7), CD4 (SK3), CD8 (SK1), CD11a (HI111), CD29 (TS2/16), CD34 (581), CD44 (Bu52), CD45 (HI30), CD49d (9F10), CD49e (IIA1), CD49f (GoH3), CD56 (B159), CD69 (L78), CD133 (293C3), CD162 (KPL-1), CXCR4 (12G5), CXCR7 (358426), NKp44 (P44-8) and β7 integrin (12G5). Cell viability was assessed using Annexin V and 7AAD (BD Biosciences). For cell cycle analysis, cells were fixed with 70% Ethanol/30% PBS for at least 1 h at 4°C. The fixed cell pellet was then incubated for 10 min at RT with RNAse at 0.17 mg/mL. To stain the DNA, the cells were incubated for 1 h at 37°C with propidium iodide at 36 ug/mL and then analyzed by flow cytometry.

Detection of apoptosis was determined by using Annexin V-7AAD staining. Caski and SiHa cervical cancer cells (1 × 10⁶) were plated in a 100 mm dish and allowed to adhere overnight. The next day, cells were treated with 10, 20 and 25 μM CUR, Nano-CUR or equivalent amounts of controls for 24 hrs. At the end of indicated time, both adherent and floating cells were collected and stained with Annexin V and 7-AAD (BD Biosciences) at 5 μL of each/100 μL of cell suspension. Cells were incubated for 20 min in the dark at room temperature and analyzed with an Accuri C6 Flow Cytometer in FL2 and FL3 channels.