Anti cd25 pe

Femoral bone marrow, mesenteric and liver (celiac, portal) lymph nodes cells were isolated, washed, and counted. Cells were treated with FcR-block (Miltenyi Biotec), surface stains were performed and intracellular stains were carried out following the fixation-permeabilization buffer manufacturer’s protocol (eBioscience). T_REGcells: anti-CD4-FITC (Miltenyi Biotec, clone GK1.5), anti-CD25-PE (eBioscience, clone PC61.5), anti-FOXP3-APC (eBioscience, clone FJK-16s). T_H17 cells: anti-CD4-FITC (Miltenyi Biotec, clone GK1.5), anti-CD196-PE (Miltenyi Biotec, clone REA277), anti- RORγt-APC (Miltenyi Biotec, clone REA278). T_H1 cells: anti-CD4-FITC (Miltenyi Biotec, clone GK1.5), anti-CD183-PE (Miltenyi Biotec, clone CXCR3-173), anti-T-bet-APC (Miltenyi Biotec, clone REA102). Dead cells were excluded via e450 viability dye (eBioscience). Data was acquired by the MACSQuant System. Analyses were performed via FlowJo VX software.

For flow cytometry analysis cells were washed with PBS containing 0.5% BSA, either surface stained with anti-CD4-eFluor-780, CD4-FITC (eBioscience), anti-CD25-PE, anti-CTLA4-PE, anti-CD39-PE, anti-Helios-PE, anti-TIGIT-PE or fixed, permeabilized, and stained with anti-, Anti-Ki67-PE, anti-Foxp3-APC and isotype controls antibodies (eBioscience) (1:100) in dark. Samples were analyzed using CyAn ADP Analyzer (Beckman and Coulter) and data analysis was done using Summit v4.3 software.

Peripheral blood mononuclear cells (PBMCs) were isolated using Lymphoprep (Axis Shield, Oslo, Norway) centrifugation and stored in liquid nitrogen until further use. Cells were defrozen and subsequently fixed and permeabilized using the Foxp3 / Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA, USA) and stained for flow cytometric analysis with anti-CD3-PerCP (BD Biosciences, San Jose, CA, USA), anti-CD8-APC-eFluor780 (eBioscience), anti-CD127-Alexa647 (Biolegend, San Diego, CA, USA), anti-CD25-PE (eBioscience), anti-CD45RO-Alexa700 (Biolegend), anti-CD45RA-Alexa605 (BD Biosciences) and anti-FoxP3-Alexa488 (eBioscience). Cells were measured on an LSR-II flow cytometer (BD Biosciences) and the data were analyzed with Kaluza 1.2 Analysis Software (Beckman Coulter, Woerden, The Netherlands).
Absolute numbers of lymphocytes in EDTA anti-coagulated blood were determined using the BD MultiTest TruCount method with sixcolor MultiTest reagents detecting CD45, CD3, CD4, CD8, CD19, CD16 and CD56 (BD Biosciences) according to the manufacturer’s instructions. Percentages of (functional) Treg subsets were characterized as described before [7 (link),16 (link)] and CD4+ T-cell counts were used to calculate the absolute numbers of these subsets.

Blood was sampled from the retro-orbital sinus of 15 mice once per month for 8 time points (total of 120 samples). Mononuclear cells from the peripheral blood was isolated by density gradient centrifugation using Ficoll (Ficoll PaqueTM plus, GE Health Care), Single cell suspensions were prepared from thymus and spleen that were removed from each mouse at the end of the experiment. For cell sorting, cells were stained with the following fluorescently labeled monoclonal antibodies: anti-CD4 Pacific Blue (BD), anti-CD25 PE (eBioscience), anti-CD44 APC (BD) and anti-CD62L PE-Cy7 (eBioscience) and viability using the Fixable Viability stain 450 (BD Horizon). Cell sorting was performed using FACS ARIA III sorter. CD4+ D44loCD62Lhi were sorted as naive T cells. After sorting, cells were pelleted and resuspended with 300μl of RNA protect cell reagent (Qiagen). Cells were stored at minus 80°C until RNA extraction. RNA was purified from RNAprotect-stabilized cells using the RNeasy Plus Mini Kit. After RNA extraction, samples were run on TapeStation to estimate quality.

The spinal cord, dissected from mice transcardially perfused with PBS, was minced into 1 mm³ pieces in solution containing 1 mg/mL or 0.1 mg/mL collagenase IV (Worthington Biochemical Corporation, USA) plus 0.4 mg/mL DNase I (Roche, Switzerland), and incubated at 37 °C for 15 min. For isolation of immune cells and microglia, cells were resuspended in 37% Percoll (GE Healthcare, USA) and centrifuged at 780 × g for 20 min. After centrifugation, myelin debris was removed and the cell pellet was collected. Cells were incubated with anti-CD16/CD32 antibodies (eBioscience, USA) for blocking Fc receptors, and then stained with combinations of the following antibodies: anti-CD45-PE-Cy7, anti-CD45-APC-Cy7, anti-CD4-PE, anti-CD8a-FITC, anti-CD8a-APC-Cy7, anti-NK1.1-PErCP-Cy5.5, anti-NK1.1-PE, anti-CD11c-PE, anti-CD11b-APC-Cy7, anti-CD11b-PerCP-Cy5.5 (BD Biosciences, USA), anti-CD4-APC, anti-CD3e-PerCP-Cy5.5, anti-CD86-APC, anti-CD25-PE (eBioscience, USA), anti-CD11c-APC, anti-I-A/I-E (MHC class II)-FITC, anti-CD4-PE-Cy7, anti-CD4-APC, anti-CD68-PE, anti-H-2K^b/H-2D^b (MHC class I)-PE, anti-CD206-PE, anti-CD178 (FasL)-PE, and Armenian Hamster IgG Isotype control-PE (BioLegend, USA). Flow cytometry was performed by using FACS Aria and FACS Verse flow cytometers (BD Biosciences, USA) and the data was further analyzed using FlowJo Software (FlowJo, LLC, USA).

Immune cells were stained with antibodies for cell surface and intracellular staining. Antibodies such as CD4, CD8, CD19, CD11c, CD11b, NK1.1, CD25, FoxP3 for flow cytometry were purchased from eBioscience (San Diego, CA, USA) and BD Biosciences. Characteristic markers were used to identify each lymphocyte type, as follows: anti-CD4-APC for CD4⁺ T cells, anti-CD8-PE for CD8⁺ T cells, anti-CD19-APC for B cells, anti-CD11c-APC for DCs, anti-CD11b-PE for macrophages, and anti-NK1.1-PE-Cy7 for NKs. These markers are expressed on the immune cell surface; thus, they were identified by cell surface staining. Immune cells were stained with antibodies against these cell surface markers for 30 min at 4 °C and washed for analysis on a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA). An intracellular staining technique was performed using the following stains: Tregs were identified using CD4⁺CD25⁺FoxP3⁺ markers because of the expression of FoxP3 in the cell nucleus. Briefly, immune cells were stained with anti-CD4-APC and anti-CD25-PE for 30 min at 4 °C and washed for fixation/permeabilization (eBioscience, San Diego, CA, USA) for 30 min at 4 °C. Finally, cells were stained with anti-FoxP3-PerCP for 30 min. For the analysis of Tregs, live cells were gated first, followed by gating of CD4⁺ T cells, then CD25⁺FoxP3⁺-expressing cells.