Ebioscience fixable viability dye efluor 450

The cytotoxic effect of the membrane blebs was analyzed, through an indirect cell viability analysis. Briefly, the splenocytes were cultured in a 24-well microplate with a density of 1 × 10⁶ cell/mL with supplemented media, then the splenocytes were stimulated with 1, 10, and 25 μg/mL of membrane blebs and incubated 24 h with 5% CO₂ at 37°C. The cells were harvested, washed with sterile PBS. Phytohemagglutinin (PHA) 20 μg, was used as positive control. 1 μL of eBioscience fixable viability dye eFluor 450 (Thermo Fisher-Scientific) was added, incubated 30 min in the darkness, washed with FACS buffer, fixed with PBS containing1% paraformaldehyde (PFA), and suspended in 400 μL of FACS buffer. Samples were analyzed in LSRFortessa^TM cytometer (Becton-Dickinson), 30,000 events were acquired and data were analyzed with FlowJo^® software v. 10 (FlowJo, LLC, Ashland, Ore).

Peripheral blood and spleens were collected 14 days after tumor implantation. Spleens were pressed into single-cell through a 70 μm cell strainer (251200, Sorfa, China), followed by removal of the red blood cells with RBC lysis buffer (00430054, ThermoFisher, USA). Cells were incubated for 10 minutes on ice with anti-mouse CD16/32 antibody (14–0161–82, Thermo Fisher, USA) for Fc blocking. For CD4⁺ and CD8⁺ T-cell detection, cells were stained with eBioscience™ Fixable Viability Dye eFluor™ 450 (65–0863–14, Thermo Fisher, USA), anti-CD45 (63–0451–82, Super Bright™ 600 rat anti-mouse CD45, Thermo Fisher, USA), anti-CD3 (11–0031–82, FITC rat anti-mouse CD3, Thermo Fisher, USA), anti-CD4 (17–0042–82, APC rat anti-Mouse CD4, Thermo Fisher, USA), and anti-CD8 (45–0081–80, PerCP-Cyanine5.5 rat anti-mouse, Thermo Fisher, USA) antibodies for 30 minutes at 4°C. Blood samples were stained directly, then mixed with 1 mL of RBC lysis buffer and incubated at 37°C for 5 minutes so that red blood cells could be lysed. Sample data were acquired with LSRFortessa (BD bioscience, NJ, USA) and analyzed with FlowJo software (FlowJo, Ashland, OR, USA).

Approximately 1 x 10⁶ RBL SX-38 cells/condition were sensitized overnight with peanut IgE mAbs (500 ng/mL each) or Der p 2 IgE mAbs (500 ng/mL each) in the presence or absence of 250 nM omalizumab (Genentech, San Francisco, CA and Novartis, Basel, Switzerland). Cells were then washed and stained with eBioscience Fixable Viability Dye eFluor 450 (Thermofisher) and human IgE clone MHE-18-Alexa Fluor 647 (BioLegend, San Diego, CA). Cells were washed again, fixed, and acquired on an Attune NxT acoustic focusing cytometer (Life Technologies-Thermofisher). Flow cytometry data were analyzed using FlowJo software (Becton, Dickinson & Company, Ashland, OR).