Anti cd4 af488

Mouse cells were stained with the following antibodies: anti-CD8-Pacific Blue, anti-CD4-APC, anti-CD11b-PE (cl. M1/70), anti-CD11c-APC. Propidium iodide was used to exclude dead cells. Data was acquired using a FACS CantoII, LSR II, or LSRFortessa (BD Biosciences) and analyzed using FlowJo (Tree Star, Ashland, OR).
Human cells were stained with the following antibodies: anti-CD3-APC (cl.OKT3), anti-CD45RA-PE-Cy7 (cl. HI100) and anti-CD4-AF488 (cl.OKT4) from Biolegend (San Diego, CA) and an anti-DC-SIGN-APC (cl.9E9A8) (R & D Systems, Minneapolis, MN). Dead cells were excluded using Sytox blue and 7-AAD (7-Aminoactinomycin D) dead stain dye (Invitrogen). Data were acquired using a FACS CantoII (BD Biosciences) and analyzed using FlowJo (Tree Star, Ashland, OR).

We used flow cytometry to verify the proliferation and differentiation of the Th17 cells and Treg cells in the blood. Blood samples were drawn into heparinized tubes and immediately used in the experiment. CD3+CD4+CD8a—IL-17+Th17 cells: cells were stimulated for four hours with Leuko Actvtn Cktl with Glgplg (BD Pharmingen) and RPMI Medium Modified (HyClone) in a constant temperature box at 37°C. Following the instructions of the IntraSureTMKit (BD Pharmingen), RBC Lysis Buffer (CWBIO) was added, and intracellular Foxp3 was stained. CD4+CD25+Foxp3+Treg cells: RBC Lysis Buffer (CWBIO) was added to the blood and washed with PBS, and the surface markers were stained with the corresponding fluorescent-labeled antibodies for 20 min. Intracellular staining of IL-17 was performed using the Transcription Factor Buffer Set (BD Pharmingen). The following antibodies were purchased from BD Pharmingen or Biolegend: anti-CD25-PE, anti-Foxp3-APC, anti-CD4-AF488, anti-CD3-APC, anti-CD8a-PerCP, and anti-IL-17-PE. Flow cytometric data were obtained using a FACSCalibur Flow Cytometer system (BD Pharmingen) and analyzed using FlowJo software.