Anti mouse cd45 bv510

For Treg/Teff cell staining (Yang, et al., 2018 (link)), live/dead cells were first labeled with antibodies to distinguish living cells (Waltham, Ma, United States). The antibodies used included; anti mouse CD4 percp-cy5.5, anti-mouse cd45-bv510, anti-mouse IL-17A APC and anti-mouse IFN-γ PE, anti-mouse TNF-α Bv421, anti-mouse Foxp3 FITC (all from BioLegend). For Teff cells, cytokine staining analysis mainly containing Th17 and Th1, the cells mixed with acetate (50 ng/ml; Sigma-Aldrich), ionomycin (500 ng/ml; Sigma-Aldrich) and brefidine (1 µg/ml; Sigma-Aldrich) were cultured together and placed in 96 well plates with cytokine secretion blockers for 4 h. Stained cells were measured using BD LSR Fortessa flow cytometer (BD Biosciences) and data were obtained and were analyzed with Flowjo 10.0 (Flowjo company, United States).

To test the CD4+ lymphocytes, LIVE/DEAD (Thermo Fisher Scientific, Waltham, MA, USA) was first labeled to distinguish the living cells. Surface marker antimouse CD4 and CD45 were labeled to all the cultured cells. After further fixation and permeabilization were performed for intracellular staining, intracellcular inflammatory cytokines were labeled and evaluated using an LSRFortessa (BD Biosciences). The following antimouse antibodies used for the cytometry analysis were purchased from BioLegend (San Diego, CA, USA) or Abcam (Cambridge, MA, USA) including antimouse CD4 Percp-cy5.5 (Biolegend), antimouse CD45 BV510 (Biolegend), antimouse IL17A APC (Biolegend), antimouse IFN-γ PE (Biolegend), antimouse TNFα BV421 (Biolegend), antimouse Foxp3 FITC (Biolegend), antimouse pPI3K PE (Biolegend), antimouse pAKT APC (Biolegend), antimouse FoxO1 (Abcam), and antimouse pFoxO1 (Abcam). SC79 (Selleck Chemicals, Houston, TX), as an AKT activator, was also used to stimulate the cells isolated from the DLNs and spleens of the EAU group to further determine whether the PI3K/AKT/FoxO1 pathway was involved in the mechanism of apremilast treatment on EAU. All the figures and data were collected and analyzed with FlowJo software (Tree Star, Ashland, OR).

Tumor tissues of mice were cut into small pieces and lysed using collagenase IV (1 mg/mL, Sigma, USA) and DNase I (Invitrogen, USA) for 1 h at 37 °C. Then, we filtrated the tissue medium using a 70 μm filter to obtain single-cell suspensions. The cell suspensions were stained with antibodies for 30 minutes on ice and subjected to FCM analysis. The following reagents and antibodies were used: LIVE/DEAD™ Fixable Stain (Invitrogen, California, USA), anti-mouse CD45-BV510 (Biolegend, California, USA), anti-mouse CD3-BUV395 (BD, New Jersey, USA), anti-mouse CD4-FITC (BD, New Jersey, USA), anti-mouse CD8-PerCP-Cy5.5 (BD, New Jersey, USA), anti-mouse FOXP3-PE (Biolegend, California, USA), anti-mouse CD25-PE-cy7 (Biolegend, California, USA), anti-mouse CD127-BV711 (BD, New Jersey, USA ).