Sp6800 spectral analyzer

Cell sortings and some of the experiments were performed on FACSAria II SORP system (BD Biosciences) equipped with five lasers (excitation wavelengths: 355, 405, 488, 561 and 639 nm, respectively). For all sortings, a 100-μm nozzle (20 psi) was used, and post sorting purity was analyzed immediately after sorting. We used four additional flow cytometers to conduct the experiments: FACSVerse (BD Biosciences), Attune (1st generation, TFS), FACSCalibur (BD Biosciences), and SP6800 spectral analyzer (SONY). The advantage of including spectral analyzer on the top of conventional flow cytometers is that it allows for more detailed spectral detection. SP6800 contains similar laser excitation sources, and we used 32 channels with narrow bandpasses starting at 420 and ending at 800 nm for detection (see Sup. Table 1 for details). A specific feature of this system is its ability to calculate a so-called virtual parameter that collects the signal from all 32 channels. Furthermore, the analyzer allows to apply a spectral unmixing algorithm, detecting signal of the other fluorescent markers in the panel. Spectral unmixing is calculated with the signal previously collected from individually stained controls over the entire 420–800 nm spectrum range. Details about the instruments’ configuration are shown in Supplementary Table 1.

Tumors were collected and processed as above for scRNA-seq and stained with flow cytometry antibodies for 20 min at 4°C. Cells were washed once with PBS and fixed with 1% formalin in PBS before analysis, which was performed on a Sony Biotechnology SP6800 Spectral Analyzer and analyzed with the Sony Biotechnology SP6800 Software and FlowJo (Tree Star, Ashland, OR). Flow cytometry antibodies used in this study were purchased from BioLegend: anti-CD11b (clone M1/70), anti-CD11c (clone N418), anti-CD4 (clone GK1.5), anti-CD45 (clone 30-F11), anti-CD8 (clone 53-6.7), anti-Gr1 (clone RB6-8C5), and anti-Ly6C (clone HK1.4).

The percent of HepG2 and A431 cells incorporated with TiO₂ PEG NPs was assessed depending on changes in light scattering using flow cytometry. Forward scatter (FSC) is the laser light scattered at narrow angles to the axis of the laser beam and is proportional to the cell size. Side scatter (SSC) is the laser light scattered at a 90° angle to the axis of the laser and is proportional to the intracellular granularity, which is increased by the uptake of nanoparticles. Briefly, 10⁶ cells/well were seeded in 24 well plates and incubated at 37 °C and 5% CO₂ for 24 h, then the cells were exposed to different concentrations (0, 10, 40, 100, 400 µg/mL medium) of 100 nm, 200 nm and 300 nm TiO₂ PEG NPs. After 24 h, the cells were washed twice, collected by trypsinization, washed three times with PBS, dispersed in 1 mL of 6% heated fetal bovine serum in phosphate buffer saline (HFBS/PBS) solution and stored on ice and analyzed within one hour. Immediately prior to analysis, the cells were passed through a nylon mesh (Cell Strainer Snap Cap, Falco, NY, USA), then cellular internal granularity was assessed using SSC and cell size was assessed using FSC using a SP6800 spectral analyzer (Sony Biotechnology, Tokyo, Japan). The percentage of cells containing nanoparticles was calculated based on changes in the gated areas compared with control untreated cells.

Cellular uptake of TiO₂ PEG NPs and EGF-TiO₂ PEG NPs by A431 cells was assessed by changes in light scattering using the flow cytometric light scatter analysis [23 (link)]. Briefly, 1 × 10⁶ cells/well were seeded in 24-well plates and incubated at 37 °C and 5% CO₂ for 24 h, then the cells were exposed to TiO₂ PEG NPs and EGF-TiO₂ PEG NPs in two concentrations (10 and 100 μg/mL medium) for 24 h. Then, cells were washed twice, collected by trypsinization, washed three times with PBS, dispersed in 1 mL of 6% heated fetal bovine serum in a phosphate buffer saline (HFBS/PBS) solution, and stored on ice to be analyzed within 1 h. Immediately prior to the analysis, the cells were passed through a nylon mesh (Cell Strainer Snap Cap, Falco, NY, USA), then cellular internal granularity was assessed using side-scattered light and cell size was assessed using forward-scattered light using a SP6800 spectral analyzer (Sony Biotechnology, Tokyo, Japan). The percentage of cells incorporated with nanoparticles was calculated based on changes in the gated areas compared with a control untreated population.

Blood samples from mice were depleted of red blood cells by washing with ACK lysis buffer. They were then stained with the following fluorophore-conjugated antibodies: anti-CD8 APC (clone 53-6.7, #100712; BioLegend) and anti-CD45.1 FITC (clone A20, #110706; BioLegend). For the day 18 timepoint after tumor challenge, a larger antibody panel was used: anti-CD8 PacificBlue (clone 53-6.7, #100725; BioLegend), anti-CD45.1 FITC (clone A20, #110706; BioLegend), anti-IL7Rα APC (clone A7R34, #135012; BioLegend), and anti-CX3CR1 PE (clone SA011F11, #149006; BioLegend). Antibodies were diluted in a staining buffer consisting of 2% heat-inactivated FBS in PBS, and staining was done at 4°C for 20 min. Samples were then analyzed on a Sony SP6800 Spectral Analyzer. Gating and analysis of flow cytometry data was done in FlowJo 10.8.1. Samples that yielded fewer than 200 CD8 cells were discarded from analyses requiring the estimation of the circulating frequency of CD8⁺ CD45.1⁺ cells.

Tumors were processed for flow cytometry as above for scRNA-seq: manual dissociation, enzyme digest, filtering, followed by staining with flow cytometry antibodies as below. Peripheral blood or spleens were collected and ACK-lysed prior to staining with flow cytometry antibodies for 20 min at 4^◦C. Cells were washed once with PBS and fixed with 1% formalin in PBS before analysis, which was performed on a Sony Biotechnology SP6800 Spectral Analyzer and analyzed with the Sony Biotechnology SP6800 Software and FlowJo (Tree Star, Ashland, OR). Flow cytometry antibodies used in this study were purchased from Biolegend:, anti-CD11b (clone M1/70), anti-CD11c (clone N418), anti-CD4 (clone GK1.5), anti-CD45 (clone 30-F11), anti-CD8 (clone 53–6.7), anti-Gr1 (clone RB6–8C5), anti-Ly6C (clone HK1.4), anti-Tbet (clone 4B10), anti-TIM3 (clone RMT3–23), anti-CD111(Nectin-1) (clone R1.302), anti-CD274 (PD-L1) (clone 10F.9G2), anti-IA/IE (clone M5/114.15.2), anti-Kb/Db (clone 28–8-6), anti-NK1.1 (clone PK136), and ZombieNIR (#423105). Intracellular staining for T-bet was performed with eBioscience Foxp3/Transcription Factor Staining Buffer Set (Invitrogen 00–5523-00).

To avoid artifacts associated with dye modification of the size of NPs, we used original NPs not exposed to dye to assess cellular uptake by HepG2 cells and determine the percentage of cells containing NPs.[21 ] Briefly, 2 ml of a HepG2 cell suspension at a density of 5 × 10⁵ cells/well was seeded in a six-well plate and cultured for 24 h. The attached cells were exposed to TiO₂ and TiO₂-PEG NPs at 0, 100, 200, 400, 600, and 800 μg ml^–1. Ultrasonic processing of the NP suspensions for 30 min before and after dilution was employed to ensure adequate dispersion. After 24 h of incubation, cells were trypsinized and passed through a nylon mesh (Cell Strainer Snap Cap) after washing twice with PBS to remove excess NPs. The cells were then collected by centrifugation and suspended in 1 ml of PBS with 6% HFBS. Subsequently, the cells were stained with 1 μl of 42 μM PI (dead cells) and 2 μl of 4.3 mM thiazole orange (all cells) (BD Cell Viability Kit, BD Biosciences, Becton, Dickinson and Co., San Jose, CA, USA). Finally, stained cells were detected using an SP6800 Spectral Analyzer (Sony Biotechnology Inc., Tokyo, Japan). Cell granularity was assessed using side-scattering (SSC) light, and cell size was assessed using forward scattering light.