Cellcarrier 96 well plate

Macrophage precursors were seeded at 4 × 10⁴ cell/well in optically-clear bottom CellCarrier 96-well plates (Perkin Elmer), and differentiated in macrophage media for a week. For detection of pSYK, the pSYK/tSYK HTRF kits (Cisbio) were used, according to manufacturer’s instructions. Cells were stimulated with goat polyclonal human TREM2 antibody (AF1828, R&D systems) for 5 min at 37 °C. Normal goat IgG (R&D Systems) was used as control. Medium was aspirated and cells were lysed in supplemented lysis buffer (Cisbio), placed on an orbital shaker for 20 min at room temperature. Experiments were run with 3 replicate wells. Cell lysates were dispensed into a ProxiPlate-384 Plus (Perkin-Elmer), followed by pre-mixed Eu³⁺-cryptate and d2 antibody diluted in detection buffer. The plate was sealed and incubated overnight at RT, then read on a PHERAstar FSX (BMG Labtech) using a HTRF optic to detect emission at 665 nm and 620 nm. Data was exported as two RFU values, and signal/noise ratio was calculated according to manufacturer’s instructions.

Macrophage precursors were seeded at 3 × 10⁴ cell/well in optically-clear bottom CellCarrier 96-well plates (Perkin Elmer), and differentiated in macrophage media for a week. Cells were fixed with 4% PFA, and In situ hybridization (ISH, RNAScope Multiplex Fluorescent v2 323,110, ACD Biotechne) was carried out according to manufacturer’s instructions with catalogue probe Hs-PLCG1 472,801. ISH was followed by immunocytochemistry with anti-Iba1 (1:500, Q08578, Alpha Laboratories). Images were taken on a high content Imaging system (Operetta, Perkin Elmer) and analysed using Harmony Software (Perkin Elmer) to quantify the number of dots (RNASCope reaction products) per cell.

Cells and EVs were immobilised onto CellCarrier 96 well plates (Perkin Elmer; Bucks, UK; Cat 6005550), fixed with 3.7% paraformaldehyde, permeabilised with 0.2% Triton X in PBS and probed using anti-human CD63 antibody (Clone: H5C6, Biolegend UK, London, UK; Cat: 353005) directly conjugated to FITC and counterstained for polymerised actin using 0.2 × Alexa Fluor 555 Phalloidin (Fisher Scientific, Leicestershire, UK; cat A34055) and 300 nM DAPI (Biolegend UK, London, UK, 422801). Images were captured using the Perkin Elmer Operetta system (Perkin Elmer; Bucks, UK) at ×40 magnification.

Approximately 1 × 10⁴ NCI-H446, NCI-H520, A549, and L132 cells were seeded per well of a CellCarrier 96-well plate (PerkinElmer, Waltham, MA, USA) and allowed to attain confluence in a humidified incubator under 5% CO₂ at 37°C for 24 h before treatment. Cells were washed twice with phosphate-buffered saline (PBS) and then transferred into RPMI 1640 medium containing 0.1% FBS. The cells were treated with 0, 1, 2, or 4 μM LIP for 24 h in a humidified incubator under 5% CO₂ at 37°C. The cells were then washed thrice with PBS and subsequently stained with Hoechst 33342 (1 μg/ml; Sigma-Aldrich, St. Louis, MO, USA) and propidium iodide (PI; 5 μg/ml; Sigma-Aldrich, St. Louis, MO, USA) in PBS for 15 min at room temperature (about 20°C) in the dark. After washing with PBS, the cells were imaged with the Operetta™ High-Content Imaging System (PerkinElmer, Waltham, MA, USA) at 20× magnification and analyzed using the PerkinElmer Harmony software.

The effect of DHPITO on the cell proliferation of CRC cells was approximated using a BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594 (cat no. C0078S; Beyotime Institute of Biotechnology) while precisely following the manufacturer’s instructions. Briefly, cells were harvested, counted, seeded into the CellCarrier™-96-well plate (PerkinElmer, Inc., Waltham, USA) at a density of 1 × 10⁴ cells per well, and cultured overnight. Cells were treated with DHPITO for 24 h at concentrations of 0, 10, 20 or 40 μM; subsequently, the cells were incubated with 10 μM EdU working solution for 4 h at 37 °C. After fixation with 4% paraformaldehyde for 30 min at room temperature, the cells were blocked with QuickBlock™ Blocking Buffer for Immunol Staining (cat no. P0260; Beyotime Institute of Biotechnology, Shanghai, China) for 1 h at 37 °C. Thereafter, the cells were incubated in a click reaction buffer for EdU staining for 30 min in a dark environment before being stained with Hoechst 33342 for 10 min. Finally, the images were captured and approximated using an Operetta CLS High Content analytical system (PerkinElmer, Inc., Waltham, MA, USA).

LN229 and U87 cells were seeded in the CellCarrier™-96-well plate (PerkinElmer, Waltham, MA, USA) at a density of 5 × 10³ cells per well. After being incubated for 24 h, cells were treated with compound-7g at various concentrations. Cells were fixed with 4% paraformaldehyde for 25 min at room temperature, and then blocked for 30 min at 37 °C using QuickBlock™ Blocking Buffer for Immunol Staining (Beyotime, P0260, Shanghai, China). The cells were incubated with anti-E2F1 or anti-LC3B for 2 h at room temperature. Then, cells were incubated with Alexa Fluor 488-conjugated anti-rabbit antibody for 1 h (Thermo, A32731, Waltham, MA, USA) at room temperature, and then incubated with DAPI for 15 min at room temperature. The samples were analyzed by the High Content analysis system.