Anti cd3e percp cy5

The DRAG targeting construct was linearized using PvuI and electroporated into IB10 E14 129/ola ES cells. Stable transfectants were selected with puromycin, and resistant clones were picked and expanded. Correct integration was determined by Southern blotting, using a probe directed against the 5′ Rosa26 homology arm. Two independent ES cell clones were injected into C57Bl/6 blastocysts to generate 28 transgene-positive chimeric mice and establish two independent DRAG transgenic lines (DRAG1 and DRAG2). GFP expression in peripheral blood B and T cells was screened using anti-CD19-PE (BD, clone 1D3, dilution 1/100), anti-CD3e-PerCP-Cy5.5 (eBioscience, clone 145-2C11, 1/100), and anti-CD11b-APC (BD, clone M1/70, 1/100), and the DRAG1 line was selected. DRAG1 mice were crossed with B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J (CAGGCre-ER^TM) to obtain heterozygous mice for experimental use.

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).