Cytoflex lx cytometer

Fresh liver tissues were dissociated using a gentleMACS Dissociator (Miltenyi Biotec) and mouse Liver Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach Germany). After separation and purification from the cell suspension by Percoll gradient centrifugation, liver immune cells were stained with antibodies and then subjected to a CytoFLEX LX cytometer (Beckman Coulter, CA, USA). The antibodies used in flow cytometry are listed in Table S1.

For cytokines and exhaustion markers analysis, CAR-T cells were stimulated with irradiated 8505c-TSHR cells for 24 h. The BD Cytometric Bead Array (CBA) kit were used to quantify the secretion level of IL-2, TNFα and IFN-γ of the supernatant of CAR-T cells following manufacturer’s instruction. For intracellular cytokines staining, the Golgi plug protein transport inhibitor Brefeldin A (eBioscience, San Diego, United States) was added into the cultured medium 4 h before detection. T cells were then fixed and permeabilized with Fixation and Permeabilization kit (BD Biosciences, San Jose, United States) according to manufacturer’s instruction. The following antibodies were used: FITC mouse anti-human TSHR (Santa Cruz Biotechnology, Santa Cruz, United States), Human IL-2 Flex Set (BD Biosciences), Human TNF Flex Set (BD Biosciences), Human IFN-γ Flex Set (BD Biosciences), BV421 rat anti-human IL-2 (BD Biosciences), PE-Cy7 mouse anti-human TNFα (BD Biosciences), PE-Cy7 anti-human CD279 (PD-1) (eBioscience), eFluor 450 anti-human CD223 (LAG-3) (eBioscience). Flow cytometry was performed on a CytoFLEX LX cytometer (Beckman Coulter, Brea, United States). A moflo Astrios EQ cell sorter (Beckman Coulter) was used for cell sorting. Data were analyzed with FlowJo software (FlowJo LLC, Ashland, United States).

Cells were analyzed on a BD LSRFortessa X-20 Cell Analyzer, BD LSRFortessa Cell Analyzer, or Beckman Coulter CytoFLEX LX Cytometer. For each experiment, 10,000 to 20,000 cells were analyzed. Data were analyzed with FlowJo software.

Samples were collected in 96-well V bottom plates and pelleted at 300g for 5 min. Supernatant was aspirated, and cells were washed in 200 μl of PBS per well (Thermo Fisher Scientific, 10010049). Cells were incubated for 15 min at room temperature in the dark in 50 μl of PBS with Fc Block (1:100 dilution, BD, 564220) and Zombie ultraviolet dye (1:500 dilution, BioLegend, 423108). Next, 50 μl of antibody cocktail made up at 2× working concentration in HF buffer (HBSS, Thermo Fisher Scientific, 14175103, supplemented with 2% FBS, Thermo Fisher Scientific, 12483020) was mixed 1:1 with the cell suspension. Cells were stained for 30 min on ice protected from light and washed twice with 200 μl of HF buffer per well. Samples were analyzed on a CytoFLEX LX cytometer (Beckman), compensation was performed in CytExpert v2.3, and data were gated and plotted in FlowJo v9. Antibodies used are listed in workbook S1.

Using flow cytometry, blood samples were processed with an extensive panel of human-specific monoclonal antibodies. These antibodies included anti-CD3 PerCp, anti-CD4 BV421, anti-CD8 BV605, anti-CD19 FITC, and anti-CD45 AF700, along with other antibodies targeting immune checkpoints and pertinent markers, such as anti-PD-1 APC, anti-PD-L1 PE, anti-CTLA-4 PE, anti-CD86 APC, anti-CD200 PE, and anti-CD200R APC. All of these antibodies were sourced from BioLegend (San Diego, CA, USA). For accurate gating during cytometric analysis, FMO controls were incorporated specifically for the immune checkpoints’ antibodies.
After the antibody incubation stage, red blood cells were lysed using a specific buffer from BD (Franklin Lakes, NJ, USA). This lysing solution was prepared following the manufacturer’s protocol to ensure optimal cell lysis. The post-lysis cell suspension was then washed and analyzed on the CytoFLEX LX cytometer (Beckman Coulter, Indianapolis, IN, USA). For subsequent data interpretation, Kaluza Analysis software, version 2.1, also from Beckman Coulter, was used. The CytoFLEX LX system’s consistent quality was upheld using CytoFLEX Ready to Use Daily QC Fluorospheres reagents (Beckman Coulter, Indianapolis, IN, USA). Figure 1 and Figure 2 present the results of the sample analysis.