Annexin 5 fluorescein isothiocyanate fitc

Reagents were as follows: ethylene glycol tetraacetic acid (EGTA) (Acros Organics, Geel, Belgium, 409910250), Fura-2 AM (Biotium, Kampenhout, Belgium, 50033), Annexin V-Fluorescein isothiocyanate (FITC) (Becton Dickinson, Franklin Lakes, NJ, USA, 556419), 7-aminoactinomycin D (7-AAD) (Becton Dickinson, 555815), U73122 (Enzo Life Sciences, Farmingdale, NY, USA, BML-ST391-0005), U73343 (Enzo Life Sciences, BML-ST392-0005), venetoclax (ChemieTek, Indianapolis, IN, USA, CT-A199), anti-human IgG/M (Jackson ImmunoResearch, West Grove, PA, USA, 109-006-127). The following antibodies were used: anti-IP₃R2 (Abiocode, Agoura Hills, CA, USA, R2872-3); anti-calnexin (Enzo Life Sciences, Farmingdale, NY, USA, ADI-SPA-865-D); anti-Bcl-2 (Santa Cruz Biotechnology, Dallas, TX, USA, sc7382HRP); anti-Bim (Bioké, Leiden, The Netherlands, 2819 S); anti-GAPDH (Sigma-Aldrich, St. Louis, MO, USA, G8795); anti-vinculin (Sigma-Aldrich, Munich, Germany, V9131). The sequences of the peptides used in this study were: BIRD-2 (RKKRRQRRRGGNVYTEIKCNSLLPLAAIVRV) and TAT-Ctrl (RKKRRQRRRGGSIELDDPRPR). These peptides were synthesized by LifeTein (South Plainfield, New Jersey, USA) with a purity of at least 85%. The IP₃ sponge (pEF-GSTm49-IRES-GFP) is a protein constructed from the IP₃-binding core of the type 1 IP₃R with a single amino acid substitution (R441Q) that has a very high affinity for IP₃ [29 (link)].

MH7A cells (5 × 10⁵cell/mL) after indicated transfection or treatment of 48 h were resuspended in binding buffer (1×) and subsequently stained with Annexin V-fluorescein isothiocyanate (FITC) (10 μL) and propidium iodide (PI) (10 μL) (Becton Dickinson, San Jose, CA, USA). Finally, the examination of cell apoptosis was performed using the FACS Calibur flow cytometry (Becton Dickinson).

RAW264.7 cells were left alone or cultured with IL-4 (5 ng/ml) for 48 h. Some cells were also treated with 0.1 μM MC-LR or MC-RR. The cells were harvested, washed, and resuspended in 0.5% bovine serum albumin (BSA)-phosphate-buffered saline (PBS). Next, the cells were preincubated with mouse Fc block (eBioscience, San Diego, California, United States) for 15 min and subsequently stained with an Alexa Fluor 488-conjugated anti-CD11b Ab (eBioscience) and an allophycocyanin (APC)-conjugated anti-CD206 Ab (eBioscience) for 30 min at 4°C in the dark. Flow cytometric analyses were performed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, United States) and FlowJo software. Apoptotic cells were analyzed by flow cytometry using annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining (BD Biosciences). Some cells were fixed in 70% ethanol for overnight, stained with 10 μg/ml PI, and analyzed for cell cycle distribution by flow cytometry.

Cell cycle arrest and apoptosis were assayed by flow cytometry as described previously (30 (link)). Cells were digested with trypsin first, 70% ethanol ice-cold in advance was used to fix samples overnight, cells were treated with RNase A (Sigma-Aldrich, USA) at 5 mg/ml, stained with iodide in sodium propionate, and analyzed by flow cytometry (FACSCalibur instrument and CELLQuest software, Becton Dickinson). Propidium iodide (PI) and Annexin V–fluorescein isothiocyanate (FITC; BD Pharmingen) were used in apoptosis detection. Quest software (BD Biosciences, San Jose, CA, USA) was used to analyze the result of apoptosis and cell cycle.

Drug-treated Farage cells were stained with Annexin V-fluorescein isothiocyanate (FITC) (BD Biosciences, Heidelberg, Germany) and propidium iodide (PI) (Sigma Aldrich, St. Louis, MO, USA) and analyzed by flow cytometry. Detection of early apoptotic (Annexin V-FITC+ and PI-) and late apoptotic cells (Annexin V-FITC+ and PI+) was performed using a FACS Calibur (Becton and Dickinson). Data were analyzed with CellQuest software (Becton Dickinson).

The cell viability was determined using trypan blue stain exclusion assay (Thermo Fisher Scientific, Waltham, MA, https://www.thermofisher.com), and apoptotic cells were identified using flow cytometry with Annexin V fluorescein isothiocyanate (FITC) and 7‐aminoactinomycin D (7‐AAD) (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) staining as described previously [30].