Celltrace far red

For staining, CellTrace^TM Violet, CellTrace^TM CFSE and CellTrace^TM Far Red fluorescent dyes (Thermo Fisher Scientific, Waltham, MA, United States) were prepared according to the manufacturers’ recommendations. Cultures were stained with 2 μL CellTrace^TM dye per mL culture and incubated overnight in the dark at 12°C and 200 RPM. Stained cultures were washed twice with YP medium, as remaining unbound dye molecules would bind to the amide groups in yeast extract and peptone.

Cellular proliferation was determined by the incorporation of 5-ethynyl-20deoxy-uridine (EdU) into cellular DNA using the Click-iT EdU Flow Cytometry Assay Kit (Thermo Fisher Scientific)^{19 (link)} and using CellTrace^TM Far Red (Thermo Fisher Scientific), following the manufacturer’s instructions. Cell cycle was analyzed by flow cytometry. After treatment, cells were trypsinized, centrifuged at 1000 × g for 5 min, collected and washed with ice-cold PBS. Cellular pellets were resuspended and fixed with cold 70% ethanol overnight. After another wash with PBS, the cell pellets were resuspended in 1 ml of staining solution containing propidium iodide (PI, 50 μg ml⁻¹). Finally, the cells were incubated at 37 °C for 30 min in the dark before analysis. For cell death assays, 50,000 cells/well were seeded and treated with 1 µM staurosporine (STS), 120 µM hydrogen peroxide (H₂O₂) or 1 µM tamoxifen as indicated. Cell death was determined by flow cytometry after staining with annexin V and 7-AAD (Annexin V Apoptosis Detection Kit I, BD Pharmingen^TM)^{19 (link)}. The intracellular production of hydrogen peroxide was monitored by flow cytometry using 10 μM 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCFH2-DA) (Thermo Fisher Scientific)^{19 (link)}. Cells were analyzed in a BD FACScan. For each analysis, 10,000 events were recorded. Data were analyzed in FlowJo software v10.6.2.

The target cells (SH-SY5Y and LAN-1) were stained with CellTrace^TM Far Red using a cell proliferation kit (ThermoFisher Scientific, Waltham, MA, USA). Labeled tumor cells were subjected to PBNP-PTT at various thermal doses and immediately co-cultured with their corresponding T cells at a 1:1 effector:target cell ratio. Cells were cultured for 4 h, collected by centrifugation, and resuspended in equal volumes of buffer across all samples. Cells were stained with Zombie Green Fixable viability dye (Biolegend, #423112), blocked with human TruStain Fc block, and stained with CD3 (Biolegend, #300326) to distinguish T cells. Flow cytometry was conducted in duplicate in a 96-well plate, whereupon equal volume was collected from each sample and analyzed in the flow cytometer. T cell-mediated cytotoxicity was calculated by determining the number of live target cells (gating CellTrace Far Red positive and Zombie green negative populations). These values were normalized to the number of live target cells after respective treatments in the absence of T cells. The samples were run on a Cytoflex flow cytometer (Beckman Coulter, Brea, CA, USA).

Cytotoxicity of CD19 CAR-T cells was measured by Luciferase assay. Briefly, ffLuc expressing target cells (K562, CD19 expressing K562, and NALM6) were resuspended in RPMI medium supplemented with 10% FBS in 96-well tissue culture plates. CAR-T cells were added at varying effector to target cell ratios. After 24-h incubation, cells were lysed in lysis reagent (Promega). Luminescence of the lysates was analyzed using a plated spectrophotometer. Spontaneous release (no CAR-T cells added) and maximum release (treated with lysis reagent) were set up. The percentage of specific lysis was calculated according to the standard formula: % specific lysis = 100×(experimental release–spontaneous release)/(maximum release–spontaneous release).
A flow cytometry-based cytotoxicity assay was used to determine cytotoxicity of GPC3 CAR-T cells. Briefly, tumor cells were pre-stained with CellTrace^TM Far Red (Thermofisher) and cocultured with CAR-T cells for 24 h. After that, cultures were added with counting beads (Thermofisher) for normalization prior to flow cytometry analysis.

We prepared a tumor spheroid in vitro to mimic an in vivo solid tumor environment. To generate a tumor spheroid, we utilized a 96-well ultralow attachment cell culture plate (Corning). In brief, A549 tumor cells were harvested, counted, and re-suspended in the complete culture media. Then, 1 × 10⁴ cells in 100 μL media were placed in each well. After that, the plate was spun at 1200 rpm for 3 min. Then, the cells were maintained in an incubator for 7 days to allow the formation of tumor spheroids. After that, the spent media were changed, and the spheroids were treated with different samples including PBS, macrophage cells only, and the Macbots (1 × 10⁴ cells/microrobots per spheroid). For visualization under a confocal microscope system, the cells/Macbots were stained with CellTrace^TM Far Red (ThermoFisher, Waltham, MA, USA) before adding them to the spheroids. Penetration of the cells/Macbots into the spheroids was allowed for 12 h in an incubator at 37 °C. After 3 washes with PBS, the spheroids were fixed with 1% paraformaldehyde for 10 min, followed by a counterstain with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI, 1 μg/mL). Finally, the spheroids were rinsed thrice with PBS, placed in a confocal dish, and observed under a confocal microscope system.

Target cells were labeled with CellTraceTM Far Red (ThermoFisher, C34564). Adherent target cells were seeded in a 96-well plate at a density of 4,000 cells/well 24 h before addition of IncuCyte Caspase-3/7 Green Apoptosis Assay Reagent (Essen Bioscience, 4440) to each well-diluted by a factor of 1,000. Non-adherent target cells were seeded in fibronectin coated 96-well plates at a density of 30,000 to 50,000 cells/well and further incubated at room temperature for 30 min before the addition of IncuCyte Caspase-3/7 Green Apoptosis Assay Reagent. After incubation, NK cells were added at various E:T ratios and monitored on the IncuCyte ZOOM to acquire images every 1 h for adherent cells and every 30 min for non-adherent cells. Experiments were performed with 3 independent biological triplicates. The cytotoxicity of target cells was analyzed by quantifying red cell number and/or overlay of Caspase 3/7 (green) within the red cells.