Fortessa platform

For cell death assay, RAW264.7s, THP-1s, LLCs, A549s was seeded into 6-well plates and treated with PP VII or different CMs for 24 h followed by harvested for staining. Apoptosis Detection Kit (556547, BD Biosciences) was performed as manufacturer’s recommendation. Cells were incubated with FITC-Annexin V in a buffer containing propidium iodide (PI) then processed by FACSVerse (BD Biosciences). For RAW264.7s staining, single-cell suspensions were prepared and stained according to standard protocol with Brilliant Violet 421 anti-mouse CD86 (105031, Biolegend) and APC anti-mouse F4/80 (123116, Biolegend). The data were acquired on a Fortessa Platform (BD Biosciences) and analyzed with FlowJo software (Version V10.0.7, Tree Star).

The collected tumor blocks were firstly minced into pieces and single-cell suspension were obtained by passing the tumor mince through a cell strainer (Corning, USA). After fixation and permeabilization using BD Cytofix/Cytoperm kit (554714, BD Biosciences), cells were stained with anti-CD3 (100246, Biolegend), anti-CD4 (100526, Biolegend), anti-CD45 (101917, Biolegend), or anti-CD8 (100722, Biolegend) antibody following standard flow cytometry protocol. Results were then obtained using Fortessa platform (BD Biosciences) and analyzed using FlowJo software (BD Biosciences). CD3 and CD45 were used to analyze total T cells (both CD45 and CD3 positive). Cytotoxic T cells were further analyzed using CD4 and CD8 after gating of CD3⁺ cells, while cells from inguinal lymph node were used as reference of gating.

Cell apoptosis was assessed using annexin V–fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Beyotime). Transfected A549 or H1299 cells were collected at a concentration of 2 × 10⁵ cells per well, received treatment with trypsin, and underwent resuspension with 400 μL PBS. Next, the cells were stained with 5 μL annexin V–FITC and 5 μL PI at room temperature under dark conditions for 15 min. After washing, the cells were analyzed with the use of a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA).
PBMCs that had been collected were fixed and permeated using BD Cytofix/Cytoperm kit (BD Biosciences). Following the standard procedures of flow cytometry, cells were stained with anti-CD3 (ab16669, Abcam, Cambridge, MA, USA), anti-CD45 (ab40763, Abcam), or anti-CD8 (ab237709, Abcam). The results were obtained from the Fortessa platform (BD Biosciences) and analyzed using FlowJo software (BD Biosciences). With CD45 expression, size, and granularity (forward scatter versus side scatter) serving as references for gating, CD8⁺ T cells were further analyzed using CD3 and CD8.