96 well u bottom plate

Biofilms were formed in 96-well plates as previously described^{12 (link),62 (link)}. Briefly, C. glabrata was resuspended in RPMI-MOPS at a concentration of 1.5 × 10⁶ cell/ml and 200 µl was added to each well and plates were incubated for 24 h at 37 °C. Biofilm burden was estimated using an XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) assay as previously described, but with the electron-coupling agent menadione^{62 (link)}. One hundred µl of the XTT working solution (0.75 mg/ml XTT in PBS with 10 µM menadione (from 10 mM stock in acetone)) was added to each well. After a 30 min incubation, samples were transferred to a Falcon 96 well U bottom plate and centrifuged for 3 minutes at 1,200 × g to pellet cells. Supernatants (110 µl) were collected and transferred to a 96-well flat bottom plate for absorption reading at 492 nm. To determine an equivalent burden of planktonic organisms, biofilm XTT values were compared to XTT values obtained for a dilution series of planktonic organisms. Results showed a burden of approximately 3 × 10⁶ planktonic cells/well to be similar to the biofilm burden (Supplementary Fig. 1). Therefore, this number of planktonic cells was used in neutrophil co-culture experiments comparing their response to biofilm and planktonic cells.

Human NK cells were isolated from eight healthy donors after informed consent. Briefly, buffy coats were diluted 1:2 with PBS and centrifuged under a density gradient in Lymphosep (C. C. Pro). NK cells were isolated by magnetic‐activated cell sorting according to the manufacturer's instructions (Miltenyi Biotec Bergisch) using a negative selection antibody cocktail and cultured on a 96‐well U‐bottom plate (Falcon, Corning Brand) with complete RPMI‐1640 medium (Lonza), supplemented with 5% human serum (C. C. Pro) and 100 U IL‐2 (PeproTech). The cells were maintained overnight at 37°C in 5% CO₂ at density of 1 × 10⁶ cells/mL. ICCs‐derived cells from four animals per group were cultured with isolated NK cells (1:2 and 1:5 T:E ratio) on a 96‐well U‐bottom plate (Falcon) for 4 hour at 37°C. Supernatant was collected, and cells were stained with CD3‐PerCP, CD56‐AF647 and CD107a‐PE (Biolegend) for FACS analysis. SLA‐silenced samples (shβ2M and shCIITA) were compared against shNS and NT.

CD3 T cells were isolated from four healthy donors after informed consent. Briefly, buffy coats were diluted 1:2 with phosphate buffered saline (PBS) and centrifuged under a density gradient in Lymphosep (C. C. Pro). T cells were then isolated by magnetic‐activated cell sorting according to the manufacturer's guidelines (Miltenyi Biotec) using a negative selection antibody cocktail. CD3 T cells were primed for 8 days with ICCs‐derived NT target cells (1:5 target:effector ratio) on a 96‐well flat‐bottom plate (TPP). On day 8, primed CD3 T cells were cultured with genetically engineered ICCs‐derived cells from 4 different animals per group (1:2 and 1:5 T:E ratio) on a 96‐well U‐bottom plate (Falcon) for 6 hour at 37°C on a humidified incubator. Supernatant was collected, and cells were stained with CD3‐PerCP and CD107a‐PE (Biolegend) for FACS analysis. SLA‐silenced samples (shβ2M and shCIITA) were compared against shNS and NT.

C-Kit-D816V-stimulated T cells were plated by limiting dilution (0.5–2.0 cells/well) and expanded in a 96-well U-bottom plate (Becton, Dickinson and Company) in AIM-V medium supplemented with 5% heat-inactivated human serum, 40 ng/mL anti-CD3 antibody (Clone UCHT 1; Becton, Dickinson and Company), and 120 IU/mL IL-2 (PeproTech, Inc.). The expanded clones were examined for IFNγ production after peptide stimulation, and a C-Kit-D816V-specific T cell clone was obtained. A single T cell from the obtained clone was sorted into a 96-well plate (4titude, Wotton, UK) by the FACSAria II cell sorter (Becton, Dickinson and Company). PIK3CA-H1047R specific T cells were enriched by IFNγ secretion assay (Miltenyi Biotec, Auburn, CA, USA), followed by sorting of a single T cell into a 96-well plate (4titude) by the FACSAria II cell sorter (Becton, Dickinson and Company).
The sequences of TCR genes of the sorted T cell clones were determined, as reported previously [33 (link)]. The cDNA of TCRα and TCRβ chain was amplified from single cells and sequenced at Eurofin Genomics K.K. (Tokyo, Japan).

Human blood was anti-coagulated with 20 U/ml pyrogen-free sodium heparin (American Pharmaceutical Partners, Inc., Schaumberg, IL, USA). All blood products were kept at room temperature and processed within 4 h from collection. Whole blood (WB) was mixed 1:1 with pre-warmed (37°C) RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) and 180 μl plated in a 96-well U-bottom plate (Becton Dickinson, Franklin Lakes, NJ, USA) containing 20 μl of 10× freshly prepared treatment, as described previously (23 (link)).