Cyan adp 9

At least 1 × 10⁴ transfected U251MG or A172 cells were rinsed with PBS, trypsinized and fixed in 70% pre-cold ethanol at 4°C overnight. Then cells were stained with 500μl PI/RNase Staining Buffer (BD, 550825) at room temperature for 15 min. Cell counts in different cell cycle phase were measured by CyAn ADP 9 colors flow cytometry (Beckman Coulter, USA) and the data were analyzed by Modfit LT 4.1v software. For apoptosis assay, 2 × 10⁵ transfected U251MG or A172 cells were trypsinized, and then co-incubated with Pharmingen™ PE Annexin V(BD, 554656) and 7-Amino-Actinomycin D (7-AAD)(BD, 559925) for 15 min at 4°C in lucifuge condition. After vortex for 15s, the ratio of apoptosis was measured by CyAn ADP 9 colors flow cytometry and the outcome were obtained via Summit V4.3 software.

For in vitro analysis, cells were treated with vehicle (0.01% DMSO) or 5 μM LCL161 and 0.01% BSA, 1 ng ml⁻¹ TNF-α, 250 U ml⁻¹ IFN-β or 0.1 MOI of VSVΔ51 for 24 hr. Cells were released from plates with enzyme-free dissociation buffer (Gibco) and stained with Zombie Green and the indicated antibodies. For analysis of tumour immune infiltrates, intracranial CT-2A tumours were mechanically dissociated, RBCs lysed in ACK lysis buffer and stained with Zombie Green and the indicated antibodies. In some cases, cells were stimulated with 5 ng ml⁻¹ PMA and 500 ng ml⁻¹ Ionomycin in the presence of Brefeldin A for 5 h, and intracellular antigens were processed using BD Cytofix/Cytoperm kit. Antibodies include Fc Block (101319, 1:500), PD-L1 (10F.9G2, 1:250), PD-L2 (TY25, 1:100), I-A/I-E (M5/114.15.2, 1:200) and H-2K^d/H-2D^d- (34-1-2S, 1:200), CD45 (30-F11, 1:300), CD3 (17A2, 1:500), CD4 (GK1.5, 1:500), CD8 (53-6.7, 1:500), PD-1 (29.1A12, 1:200), CD25 (PC61, 1:150), Gr1 (RB6-AC5, 1:200), F4/80 (BM8, 1:200), GrzB (GB11, 1:150) and IFN-γ (XMG1.2, 1:200). All antibodies were from BioLegend except for TNF-α (MP6-XT22, 1:200) and CD11b (M1/70, 1:100) where from BD Biosciences. Cells were analysed on a Cyan ADP 9 (Beckman Coulter) or BD Fortessa (BD Biosciences) and data were analysed with FlowJo (Tree Star).

All flow cytometric investigations were performed using a CyAn ADP 9 colour analyser (Beckman Coulter, Ltd, High Wycombe, UK) equipped with 405 nm, 488 nm, and 642 nm solid‐state lasers and 11 detectors in standard configuration. Summit software was used for acquisition and analysis (Beckman Coulter). The machine was calibrated with single peak alignment beads (Spherotech), checking that coefficients of variation (CVs) resided within the target range (set by the manufacturer for the CyAn ADP) for each channel prior to acquisition of samples. A minimum of 400,000 events per sample were acquired for each sample. Samples were filtered through 35 μm nylon cell strainer mesh tubes (BD Biosciences) directly prior to acquisition. For data analysis, events were first plotted as forward versus side scatter (SSC) using SSC on a log scale and a large gate was drawn excluding debris. Cells were then further plotted for CD3, CD14, or CD16b versus forward scatter area to identify CD3⁺ T cells, CD14⁺ monocytes, or CD16b⁺ neutrophils. CD3⁺, CD14⁺, or CD16b⁺ gated cells were finally plotted as forward scatter (FS linear) versus SSC on a log scale and regions were drawn to identify SSC low and hi cells based on the no particle control for CD3⁺, CD14⁺, or CD16b⁺.

Cancer cells dissociated from transplanted tumor tissues or from culture plates were counted and resuspended in 100 μl of HBSS containing 2% heat-inactivated FBS (HIFS) and 10⁵ cells. Five microliters of mouse IgG solution (1 mg/ml) was added and incubated on ice for 10 min. According to the manufacturer's recommendation, appropriate antibodies were added and incubated for 30 min on ice. Then, cells were washed twice with HIFS and resuspended in 0.2 ml of HIFS that contained 7-aminoactinomycin D (7-AAD, 1 μg/ml final concentration). Antibodies used were anti-CD44 (APC) and anti-CD24 (PE), which were purchased from BD Pharmingen (San Diego, CA, USA). Dead cells were eliminated by using viability dye 7-AAD. FACS was performed on a Cyan-ADP 9 (Beckman Coulter, Brea, CA, USA).