Software 7

Single-cell suspensions were stained with FITC anti-human CD11b and CD14 antibodies (1:20; cat. nos. 301329 and 301803, respectively; BioLegend, Inc.) for 30 min at 37°C. FITC mouse IgG1 and IgG2a antibodies (1:20; cat. nos. 400109 and 400207; BioLegend, Inc.) were used to stain single cell suspensions for 30 min at 37°C. These control antibodies were the isotype controls for CD11b and CD14, respectively, and were used as a reference in each experiment. In total, ~1×10⁵ events were collected for each sample with a Becton Dickinson FACS Calibur system (BD Biosciences) and FlowJo software 7.6.1 (FlowJo, LLC) was used for analysis.

For the analysis of apoptosis, AsPC-1 and PANC-1 cells were seeded at a density of 2×10⁵ cells per well in six-well plates. Cells of NC group were treated with 8 µl DMSO per well. After treatment with IC50 concentration of GSK343 (12.71 µmol/l for AsPC-1; 12.04 µmol/l for PANC-1) and 20 µmol/l of GSK343, cells were harvested and double stained with 5 µl Annexin V-FITC and 5 µl PI (Annexin V, FITC Apoptosis Detection kit; cat. no. AD10; Dojindo Molecular Technologies, Inc.) in the dark. For the assessment of cell cycle distribution, cells were collected and fixed in 75% ethanol overnight at 4°C after treatment with GSK343 for 48 h. Upon completion of treatment, cells were washed with PBS and resuspended in 500 µl of staining solution (Cell Cycle Staining kit; cat. no. CCS012; Hangzhou Multi Sciences Biotech Co., Ltd.) at 37°C for 30 min. Finally, apoptosis and cell cycle distribution were detected using flow cytometry (CYTOMICS FC 500; Beckman Coulter, Inc.). Flowjo software 7.6.1 (FlowJo LLC) and Modifit LT software 3.1 (Verity Software House, Inc.) were used to analyze the data of apoptosis and cell cycle.

After sacrificing the experimental mice at 72 hours after ICH, brain peri-hematoma tissue was harvested and suspended in RPMI-1640 medium. The tissue was then digested in a shaker at 37°C, rotating at 950 × g, for 5 minutes with the addition of type IV collagenase (1 mg/mL) and DNase I (50 mg/mL). Cell supernatants were then removed, resuspended, isolated by centrifugation, and washed with RPMI-1640 prior to flow cytometry. The final cell amount was equivalent in both groups, with a concentration of 2 × 10⁶/mL. Cells from brain peri-hematoma tissue were incubated with fluorescence-labeled antibodies against the surface markers IL-17–APC-Cy7 (Cat. No. 560821; BD Biosciences, San Jose, CA, USA) and TCR γδ–FITC (Cat. No. 559501; BD Biosciences). Incubations were carried out according to the manufacturer’s protocols, at 4°C for 40 minutes in the dark.
In addition, TCR γδ cells were isolated from circulating peripheral blood mononuclear cells of C57BL/6 mice. After cell suspension of peripheral blood mononuclear cells, TCR γδ–FITC antibody was added to the suspension and incubated in the dark for 40 minutes. The infiltration of the fluorescence-labeled suspension was then tested. Fluorescence-activated cell sorting analysis was performed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA), and data were analyzed using FlowJo software 7.6.1 (FlowJo LLC, Ashland, OR, USA).

DCs were blocked using 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at room temperature for 1 h and incubated with CD16 and 32 mAb (cat. nos. 2.4G2 and 565,502; 1:1,000; BD Biosciences, Franklin Lakes, NJ, USA) at 4°C for 15 min. Next, DCs were incubated in saturated PE-anti-MHC-II/CD80&86 mAb (cat. no. 565157; 1:1,000; BD Biosciences) at 4°C for 30 min. Cells were examined using a flow cytometer and were analyzed using FlowJo software 7.6.1 (FlowJo, LLC, Ashland, OR, USA).

As reported previously, the protein L has the ability to bind to the κ light chain of antibodies (48 (link)). Flow cytometry was used to confirm the expression of scFv-28Bz using the FITC-Protein-L reagent (cat no. RPL-PF141; ACROBiosystems). PBMCs (~2 million) were harvested using centrifugation at 300 × g for 10 min at 25°C, washed twice with phosphate-buffered saline (PBS) and incubated with 2 µg FITC-Protein-L protein at room temperature for 1 h in the absence of light, with FITC-Protein-L-stained mock cells serving as the isotype control. Subsequently, the cells were washed three times in PBS containing 0.5% bovine serum albumin (BSA). NCI-H358 and A549 cells (~2 million) were harvested by digestion with trypsin and washed twice with PBS. The cell lines were incubated with 2 µg phycoerythrin (PE)-anti-PD-L1 (PE anti-human CD274 Antibody; 1:10, cat no. 329706) at room temperature for 30 min, prior to being washed three times in PBS containing 0.5% BSA. The cells were analyzed with a BD FACSCalibur flow cytometer and the results were analyzed with FlowJo software 7.6.1 (FlowJo LLC, Ashland, OR, USA).

Cell cycle distribution was detected using flow cytometry. After the CRC cells had been harvested and washed, cells were detected with a Cell Cycle and Apoptosis Analysis Kit (cat. no. C1052M; Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Apoptosis was detected using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI)-Apoptosis Detection kit [MultiSciences (Lianke) Biotech Co., Ltd.]. CRC cells (5×10⁴/well) were seeded and resuspended in 12-well plates, and Annexin V-FITC (5 µl/well) and PI (100 µl/well) were added to each reaction system for 6 min. Immediately after staining, flow cytometric assays were conducted using a flow cytometer (EPICS; Beckman Coulter, Inc.). Analysis of flow cytometry results was conducted using FlowJo software7.6.1 (FlowJo, LLC).

An Annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis detection kit (MBL International Co., Woburn, MA, USA) was used to examine the percentage of apoptotic cells. In brief, 1×10⁵ cells were washed with cold PBS and re-suspended in binding buffer provided in the aforementioned kit. Cells were incubated at room temperature with 10 µl Annexin V-FITC and 5 µl PI for 15 min, and were then analyzed using a Gallios flow cytometer (Beckman Coulter, Inc.) and FlowJo software 7.6.1 (FlowJo LLC, Ashland, OR, USA).