Pma ionomycin p 1

The cross-reactivity between HDM and helminth homologue proteins was also assessed at T cell level by stimulating the inflammatory lung cells from mice chronically sensitized with house dust mite extract, with different conditions. Briefly, lung cells from HDM-allergic mice were obtained as described by [16 (link)] were incubated overnight at 37°C, 5% CO2, in the absence (media) or upon stimulation with HDM extract (10μg/mL), rDer p 10 (10μg/mL), OvTrop (10μg/mL) and finally 0.5/0.05 nM of PMA/ionomycin (P/I) (Sigma-Aldrich, USA). 10ug/mL of Brefeldin A was added in all conditions. Cells were then stained using a panel of antibodies to profile the antigen-specific effector memory CD4⁺ T cells expressing the activation marker CD40L.
Briefly, cells were stained with a Live/Dead marker (Fixable Blue-UV450). Secondly, after washing with FACS buffer (PBS, 2% FBS), antibodies to the extracellular markers CD19, TCR-b, CD44 were used. After washing, cells were then fixed with 4.2% formaldehyde (BD Cytofix, BD Biosciences) and permeabilized (Perm buffer eBiosciences), and finally stained with CD4, CD40L antibodies (flow cytometry panel in S1 Table).

Rapamycin (Rap) (Sigma, USA) was dissolved in DMSO and then diluted with RPMI medium. Chemotherapeutic drugs paclitaxel (PTX) and adriamycin (ADM) were purchased from the First Affiliated Hospital of Jinan University (Guangzhou, China). PMA/Ionomycin (P/I) were purchased from Sigma, USA. The peptides for immunogenic B16F10 and 4T1 mutations were synthesized by Sangon Biotech (Shanghai, China) accordingly previous publication (Figure S3A). Propidium iodide (PI) was purchased from BioLegend, USA. LC3B antibody was from Cell Signaling, USA. Anti-CD3, anti-CD4, anti-CD8, and anti-FOXP3 antibodies were purchased from Abcam, Cambridge, UK. Antibodies used for flow cytometry assay were as follows: anti-CD16/32 mAb (BD Biosciences, USA), Anti-CD3-PEcy5, anti-CD4-FITC, anti-CD8-FITC, anti-IFN-γ-APC, anti-TNF-α-PE, and anti-FOXP3-PE (BioLegend, USA).