Anti mouse cd4 percp cy5

Restimulated splenocytes were transferred to V-bottom plates and stained in DPBS for 10 min at RT with fixable viability dye eFluor 780 (1:5,000, eBioscience) and anti-mouse CD4 PerCp/Cy5.5 (1:200, BioLegend, SanDiego, USA, clone GK1.5) and finally analyzed on a Cytoflex S flow cytometer (Beckmann Coulter). Proliferating cells among live CD4+ T cells were identified by their reduced fluorescence on the eFluor 450 channel. Peptides were considered immunoreactive if the stimulation index (% proliferating cells stimulated with peptide/% proliferating cells without stimulation) was >2.

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).

Splenocytes suspension was adjusted to 1×10⁶ cells/100 µl staining buffer. The
cells were stained with anti-mouse CD4-PerCP-Cy5.5, CD8-Alexa Fluor 488, CD25- APC,and
Foxp3-PE (BioLegend, USA). For the analysis of regulatory CD4+ T cells, the cells were
treated by fixation/ permeabilization solution (Biolegend, USA) according to the
manufacturer’s instructions. The incubation time was 30 minutes at 4° C. All lymphocytes
subsets were analyzed by FACS Calibur instrument (Becton Dickinson, USA). The flow
cytometer data analyses were done using the FlowJo software version 7.6.1 (Tree Star,
USA).

The mouse tumor was cut into 2- to 3-mm pieces to make a single cell suspension. The tumor and spleen were mechanically macerated and passed directly through a 70-mm nylon cell filter (Corning). The cells were washed with flowing buffer (0.1% BSA in PBS) and incubated with Zombie NIRTM (Biolegend, 1:100) for 20 min at room temperature, and then cells were stained with extracellular antibodies: anti-mouse CD3 FITC, anti-mouse CD8a APC, anti-mouse CD4 PerCP/Cy5.5, anti-mouse Ly-6G/Ly-6C (Gr-1) PE/Cy7, anti-mouse CD11b PE, anti-mouse CD 279 (PD-1) Brilliant Violet 421TM or anti-mouse CD45 Brilliant Violet 510TM (Biolegend). For intracellular staining, cells were stained using the FoxP3/T-bet/IFN-γ/granzymeB (GrzmB) staining buffer set (Biolegend) for fixation and permeabilization after completion of extracellular staining according to the manufacturer’s protocol. Cells were then stained with Biolegend’s anti-mouse FoxP3 PE, anti-mouse T-bet PE/Cy7, anti-mouse GrzmB FITC or anti-mouse IFNr bright purple 510TM. All data were collected on a flow cytometer (BD Biosciences, Canto II) and analyzed using FlowJo v10 software (Tree Star, Inc.)

HLJD decoction was prepared as above and stored at − 20 °C. The following flow cytometric antibodies were obtained from Biolegend (San Diego, CA, USA): anti-mouse CD45-APC/Cy7 (Cat#157,617), anti-mouse F4/80-PE (Cat#123,110), anti-mouse CD11b-FITC (Cat#101,206), anti-mouse CD11c-PE/Cy7 (Cat#117,318), anti-mouse CD8a-FITC (Cat#155,004), anti-mouse CD4 PerCP/Cy5.5 (Cat#100,434) and anti-mouse CD3ε-PE/Cy7 (Cat#155,706). Fixable Viability Stain (FVS) 700 was purchased from BD Bioscience (San Jose, CA, USA, Cat#564,997). Primary antibody against mouse TLR7 was purchased from Proteintech (Wuhan, China), and secondary antibodies and DAPI were obtained from Servicebio (Wuhan, China). ROS staining solution (Cat#D7008) was purchased from SIGMA (St. Louis, MO, USA). Mouse anti-PD-1 antibody and anti-CTLA-4 antibody were purchased from Bioxcell (Lebanon, NH, USA).