Annexin 5 apc

B16-OVA cells were treated with 0.03 μM doxorubicin for 24 hours. Cells were harvested and stained with propidium iodide and APC Annexin V (Biolegend). Membrane phosphatidylserine exposure was evaluated by flow cytometry and is represented by the %Annexin V+PI- cells. Alternatively, cells were labelled with an Alexa Fluor 647 anti-mouse calreticulin antibody (clone ERP3924; Abcam, Cambridge, England) and incubated on ice for 30 minutes before analysis.

The extent of apoptosis in OSCC and PDL cells after treatment was determined by staining with APC-annexin V and 7-AAD (BioLegend, London, UK), because the nanoparticles were shown not to interact with these channels. The medium and detached cells were stained with 50 µL APC-annexin V (2.5 µL/mL annexin binding buffer) for 30 min after being exposed to the tested compounds for 24 h. Cells were labeled and then washed three times with annexin-binding buffer before being placed again in a staining solution containing 2.5 µL/mL 7-AAD. The FACSCanto II flow cytometer from BD Biosciences, San Jose, California, USA, was then used to analyze the cells (10,000 events per treatment). FlowJo 10 and GraphPad Prism 7 software programs were used to analyze the data (gating strategy). The cells treated with 10 µL of DMSO served as a control group in this assay (e.g., untreated group).

Phorbol 12-myristate 13-acetate (PMA), ionomycin, and Brefeldin A (BFA) were obtained from Multisciences (China). Recombinant human IL-21 (rhIL-21) was purchased from PeoproTech GmbH (Rocky Hill, CT, USA). Anti-GrB antibody and isotype control were purchased from R&D Systems (Minneapolis, MN, USA). CpG ODN 2006 was purchased from InvivoGen (San Diego, CA, USA). Anti-human IgM + IgG for B-cell receptor (BCR) stimulation and APC-Cyanine7-anti-human CD19 antibody, FITC-anti-human CD4 antibody, PerCP-Cyanine5.5-anti-human CD4 antibody, FITC-anti-human CD14 antibody, PE-anti-human-IL-21 receptor (IL-21R) antibody, PE-Cyanine7-anti-human CD25 antibody, PE-anti-human GrB antibody, FITC-anti-human IFN-gamma antibody, and eFluor^® 660-anti-human IL-17A antibody for immunostaining were purchased from eBioscience (San Diego, CA, USA). Alexa Fluor^® 700-anti-human CD3 antibody, Brilliant Violet 570-anti-human CD56 antibody, APC-annexin V, and 7-AAD viability staining solution were purchased from Biolegend (San Diego, CA, USA).

Cell lines were seeded at 500,000 cells/ml and treated with dimethyl sulfoxide (DMSO [untreated control, vehicle only]), 15 µM camptothecin (CPT), and 10 µM Nutlin-3 for 24 h. Samples were washed in 1× Annexin binding buffer and stained with APC-annexin V (BioLegend, catalog no. 640920) for 30 min to 1 h in the dark at 4°C and then washed in fluorescence-activated cell sorter (FACS) buffer (5% fetal bovine serum [FBS] in phosphate-buffered saline [PBS]) before analysis on a BD FACSCanto II.

ALDH⁺ and ALDH^– cells were identified by using an ALDEFLUOR kit (Stem Cell Technologies) by following the ALDEFLUOR protocol. Briefly, cells were suspended in ALDEFLUOR assay buffer with addition of 1.5 mM ALDH substrate and incubated at 37°C for 40 minutes. ALDH substrate was washed away with cold ALDEFLUOR buffer before analysis. Diethylaminobenzaldehyde (DEAB), which is a specific inhibitor of ALDH, was used to control for background fluorescence. Cells were analyzed by LSR Fortessa flow cytometer (BD) and sorted with a FACSAria 6-laser sorter (BD). For apoptosis analysis, cells were stained with APC Annexin V (BioLegend) by following the manufacturer’s protocol and analyzed by LSR Fortessa flow cytometer.

Single-cells suspensions were obtained from peritoneal lavage fluid, peripheral blood, and bone marrow of 6 to 10-week-old Alkbh5^–/– mice and WT littermates, then were labeled with fluorescently labeled antibodies as described previously^{15 (link)} and filtered through 40-μm cell strainers. All the samples were analyzed on LSRFortessa (BD Biosciences) and analyzed with FlowJo (TreeStar). Antibodies that used for staining cells are as following: Mouse: PE-Cy5 anti-mouse CD45 (BD Pharmingen), APC or PE-Cy7 anti-mouse CD11b (BD Biosciences), FITC anti-mouse Ly6G (BD Biosciences), PerCP or PE-Cy7 anti-mouse F4/80 (Biolegend), APC anti-mouse CXCR2 (Biolegend). Human: PE anti-human CXCR2 (Biolegend). Cell apoptosis: APC Annexin V (Biolegend), PerCP 7-AAD (Biolegend). Cells were defined as: mouse neutrophils (Ly6G⁺ CD11b⁺), mouse macrophages (F4/80⁺ CD11b⁺), and apoptotic neutrophils (Ly6G⁺ CD11b⁺ Annexin V⁺).