Computational protocol: Selective degradation of PU.1 during autophagy represses the differentiation and antitumour activity of TH9 cells

Similar protocols

Protocol publication

[…] After 72 h of polarization, cell culture supernatants were assayed by ELISA for mouse IFN-γ, IL-4, IL-5, IL-13, IL-17 and IL-9 (BioLegend) according to the manufacturer’s protocol.For intracellular staining, cells were cultured for 3 days, restimulated for 2 additional days and then stimulated for 4 h at 37 °C in culture medium containing PMA (50 ng ml−1; Sigma-Aldrich) and ionomycin (1 μg ml−1; Sigma-Aldrich). After staining for surface markers and 7-AAD, cells were fixed and permeabilized according to the manufacturer’s instructions (Cytofix/Cytoperm kit, BD Biosciences), and then stained for intracellular products. Monoclonal antibodies used for flow cytometry analyses were as follows: fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (GK1.5, BD Biosciences, dilution 1:300), PC7-conjugated CD8 (BD Biosciences, dilution 1:300), allophycocyanin (APC7)-conjugated CD45 (BD Biosciences, dilution 1:300), Brilliant Violet (BV421)-conjugated Foxp3 (BD Biosciences, dilution 1:100), phycoerythrin (PE)-conjugated IFN-γ (BD Biosciences, dilution 1:300), PE-conjugated IL-4 (BD Biosciences, dilution 1:300), APC-conjugated anti-IL-9 (RM9A4, BioLegend, dilution 1:100) and APC-conjugated IL-17A (Pharmingen, dilution 1:300). All events were acquired by a BD LSR-II cytometer equipped with BD FACSDiva software (BD Biosciences) and data were analysed using FlowJo software (Tree Star, Ashland, OR, USA). […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Application Flow cytometry
Organisms Mus musculus
Diseases Neoplasms