Annexin v 7 aad detection kit

Cell surface expression of L1CAM (clone REA163, Miltenyi Biotec), GD2 (clone 14.G2a; BD), CD3 (clone Hit3a, BioLegend, San Diego, CA, USA) and CD8 (clone SK1; BioLegend) was detected by fluorophore-conjugated monoclonal antibodies on a Fortessa X-20 (BD Biosciences, Franklin Lakes, NJ, USA) 4-laser flow cytometer. EGFRt expression was detected using biotinylated cetuximab (Bristol-Myers Squibb, New York, NY, USA) and a phycoerythrin (PE)-conjugated streptavidin antibody (cat #12-4317-87, BioLegend). Activation was assessed by fluorophore-conjugated monoclonal antibodies detecting TNFRSF9 (formerly CD137, clone 4B4-1; BioLegend) and IL2RA (formerly CD25, clone BC96; BioLegend). The Annexin V/7-AAD detection kit (BioLegend) was used to assess apoptosis. Dead cells were excluded from analyses using the LIVE/DEAD™ Fixable Green Dead Cell Stain Kit (cat#L23101, Life Technologies). Precision count beads (BioLegend) were used to quantify T cell infiltration according to the manufacturer’s instructions. Flow cytometry data was processed using FlowJo_V10 Software (Tree Star Inc., Ashland, OR, USA).

The drugs, including Cln and GCV, were purchased from Sigma-Aldrich (St. Louis, MO, USA), and ICI 118551 hydrochloride (ICI) was purchased from Tocris Bioscience (Bristol, UK). The antibodies, including rabbit monoclonal anti-S100-β, rabbit polyclonal anti-Cx43, goat anti-rabbit IgG H&L (HRP), and goat polyclonal anti-rabbit IgG H&L (Alexa Fluor 488) were bought from Abcam (Cambridge, MA, USA). The rabbit polyclonal primary antibody against cleaved caspase-3 was purchased from Cell Signaling Technology (Beverly, MA, USA), anti-Bax, anti-Bcl-2, and anti-TK were bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The rabbit polyclonal anti-β-actin was obtained from PADZA (Tehran, Iran). DMEM/F12, fetal bovine serum (FBS), antibiotic/antimycotic, trypsin-EDTA, and MTT were purchased from Gibco Life Technologies (Carlsbad, CA, USA). The Annexin V/7-AAD detection kit, TRIzol reagent, and the reverse transcription kit were procured from BioLegend (San Diego, CA, USA), Thermo Fisher Scientific (Waltham, MA, USA), and Takara (Shiga, Japan), respectively. The enhanced chemiluminescence (ECL) kit was bought from Amersham Biosciences (Freiburg, Germany).