Facs gallios

ROS production was measured with the cell-permeable probe CM-H2DCFDA. The cells were plated 24 hr before the assay in six-well plates. The CM-H2DCFDA dye was loaded by incubation at a concentration of 10 μM for 30 min at 37°C. After incubation, cells were washed twice with PBS and treated with H₂O₂. Fluorescence was quantified at 0, 15, 15, 30, 60, and 120 min of treatment with H₂O₂ using a flow cytometer (FACS Gallios; Beckman Coulter, Brea, CA) with excitation at 485 nm and emission at 530 nm. The threshold value was set at 5 min in the vector group, and subsequently, the change in threshold percentage of the fluorescence intensity of each group was analyzed.

PTC-derived cells were cultured at about 60-80% confluence in 6-well dishes in 1:1 DMEM/Ham's F12 or high glucose DMEM supplemented with 10% FBS then treated with 10 μM vemurafenib, 100 nM obatoclax, combination of 5 μM vemurafenib plus 100 nM obatoclax, or vehicle for 48 hrs in 1:1 DMEM/Ham's F12 or high glucose DMEM supplemented with 0.2% FBS. After 48 hours, adherent cells were trypsinized and pelleted with the supernatant. Cell pellets were fixed in pre-chilled (−20°C) ethanol 75% for propidium iodide staining and flow cytometry analysis a FACS Gallios (Beckman Coulter, Miami, FL, USA) to evaluate sub-G1 cell population (apoptosis).

To determine the lipid accumulation, cells were detached with TrypLE Select (Life Technologies, Carlsbad, California, USA) and stained using 12.5 ng/ml of Nile Red (Invitrogen, Carlsbad, California, USA), to mark intracellular neutral lipids. After washing with PBS to ensure the removal of unbound dye, quantitative results were obtained by evaluating Nile Red fluorescence with FACS Gallios (Beckman Coulter, Brea, California, USA).

For surface staining, 1–20 × 10⁵ cells were stained on ice, washed twice, and then measured with a FACS Gallios (Beckman Coulter). For intracellular staining, the cells were fixed with 4% PFA and permeabilized with 0.5% saponine. Data were exported in FCS‐3.0 format and analyzed with FlowJo software (TreeStar). A Bio-Rad S3e cell sorter was used to select CD20-negative cell populations. Additional details can be found in SI Appendix, Methods and Dataset S4.

H1299 cells were detached with 2 mL of 1.5 mM EDTA in PBS (-/-), and 1 × 10⁵ cells were counted and transferred to a U-bottom 96 well plate (Sarstedt AG & Co. KG, Numbrecht, Germany). To quantify protein binding to cell surface receptors, cells were incubated with different concentrations of fluorescently labeled FS-Janus lectin and CBM40 solutions for 30 min at 4 °C and protected from light compared to PBS-treated cells as a negative control. For the saturation of fucose-binding sites, 32 nM FS-Janus lectin was pre-incubated with 25, 50, or 100 mM soluble l-fucose, for 30 min at RT, in the absence of light. At the end of pre-incubation, the solution was diluted 100 times and added to cells for 30 min at 4 °C, in the dark. Subsequently, cells were centrifuged at 1600× g for 3 min at 4 °C and washed twice with FACS buffer (PBS (-/-) supplemented with 3% FCS v/v). After the last washing step, the cells were re-suspended with FACS buffer and transferred to FACS tubes (Kisker Biotech GmbH Co. KG, Steinfurt, Germany) on ice and protected from light. The fluorescence intensity of treated cells was monitored at FACS Gallios (Beckman Coulter Inc., Brea, CA, USA) and further analyzed using FlowJo V.10.5.3.

Blood was collected from the tail vein on days 0, 4, 10, 14, 20, 24, and 28 from mice during AIA and mixed with heparin to prevent coagulation. Spleens and a pool of draining lymph nodes (axillary, popliteal, and inguinal) were collected on days 0, 4, 10, 24, and 28 of AIA, from which single cell suspensions were prepared by gently crushing and passing the spleen and lymph nodes through a 70 µm nylon cell strainer and lysing the red blood cells with an RBC lysing solution (Sigma-Aldrich, Dusseldorf, Germany). The single cell suspensions or 100 µl of heparinized blood were surface stained with rat anti-mouse CD4 FITC antibody (Clone GK1.5, BD Biosciences, San Jose, CA, USA) and rat anti-mouse CD25 PE antibody (Clone PC61.5, eBioscience, San Diego, CA, USA). The cells were then fixed and permeabilized with a Foxp3-staining set (eBioscience, USA) according to the manufacturer’s instructions and stained intra-cellularly with rat anti-mouse Foxp3 APC antibody (Clone FJK-16s, eBioscience, USA). Analysis was performed with FACS Gallios (Beckman Coulter, Inc., Brea, CA, United States), and collected data were analyzed with Kaluza^® Flow Analysis Software, Beckman Coulter (version 1.5). The percentages of Foxp3⁺ T_regs among gated CD4⁺ cells were determined by FMO gating as earlier described (20 (link)).