96 well clear round bottom ultra low attachment microplate

To differentiate iPSCs into clumps of cardiomyocytes, we modified a previously reported protocol (38 (link)) to reduce the population of cardiomyocytes before purification. StemPro-34 supplemented with 2 mM l-glutamine, l-ascorbic acid (50 μg/ml), transferrin (150 μg/ml), and 0.4 μM monothioglycerol was used as the basal medium. Briefly, feeder-free iPS-MYH6-GFP cells were suspended in differentiation medium 1 [basal medium supplemented with 0.5% (v/v) Matrigel matrix, 10 μM Y-27632 (Wako), and recombinant human BMP4 (2 ng/ml)] in a 100-mm ultralow attachment culture dish or 96-well clear round-bottom ultralow attachment microplate (Corning). On day 1, an equal volume of differentiation medium 2 [basal medium supplemented with rhBMP4 (18 ng/ml), recombinant human Activin A (12 ng/ml), and recombinant human basic FGF (10 ng/ml)] was added to the dish. On day 3, the medium was replaced with differentiation medium 3 [basal medium supplemented with 0.5% (v/v) Matrigel matrix, 1 μM stemolecule Wnt Inhibitor https://www.reprocell.com/product-catalog/small-molecules/stemolecule-wnt-inhibitor-iwp-3, and recombinant human VEGF (10 ng/ml)]. On day 7, the medium was replaced with differentiation medium 4 [basal medium supplemented with rhVEGF (5 ng/ml)]. After day 10, the medium was replaced with fresh differentiation medium 4 every few days.

A549-NLR cells (5000/well) were seeded in a 96-well clear round-bottom ultra-low attachment microplate (Corning, Corning, NY, USA), centrifuged at 130× g for 10 min, and incubated for 3–4 days to form spheroids. NK cells were then added in the presence of Ultra-LEAF isotype or anti-TIGIT antibodies (Biolegend, San Diego, CA, USA). After 7 days of incubation, NK cells were stimulated with PVR⁻ or PVR⁺-K562-GFPLuc cells for 4–6 h in the presence of Brefeldin A (eBioscience, San Diego, CA, USA) and Golgi Stop™ (BD Biosciences, Franklin Lakes, NJ, USA). Samples were harvested and stained with CD56, CD3, and CD107a antibodies, fixed and permeabilized (eBioscience IC Fixation and Permeabilization buffers), and probed with antibodies for IFNγ and TNFα, followed by analysis using flow cytometry.

MKN45 cells (1000 cells in 200 μL of complete medium) were added to a 96-well clear round bottom ultra-low attachment microplate (Corning, USA). Three-dimensional (3D) tumour cell spheroids were constructed and cultured, and the experiment could be performed when the cell spheroid diameter has proliferated to about 200–300 μm. The experiment was divided into two groups, the HG-CAR-PM group, and the HF-CAR-PM group. The human peritoneal cells were transfected 1 day before the experiment, and the cells of each group were collected, centrifuged, and washed, then added complete culture medium and resuspended to 5 × 10⁴ cells/mL. Aspirate 100 μL of culture medium from each spheroids culture, add PMs from different groups at an effective target ratio of 5:1 to each culture, and incubate for 16 h, and then add tumour cells in each well. The spheres were carefully aspirated into 1.5 mL EP tubes and washed twice with saline to remove free PMs. The washed tumour cell spheroids were placed in a confocal small petri dish and fluorescence confocal microscopy was performed to observe the penetrating infiltration of PMs in the tumour cell spheres. The images were processed using Image J software 1.37.