Easysep mouse cd11c positive selection kit

Back skin tissues dissected from euthanized mice were placed into dishes and cut into 0.2mm² pieces with scissors. The pieces of tissue were transferred into 100 mm dishes and incubated with a digestion solution containing 2.5 mg/mL collagenase I (Cat # C0130, Sigma), 2.5 mg/mL trypsin (Cat # 27250018, Thermo Fisher Scientific), and 1 U/mL DNase I (D8071, Solarbio) in Roswell Park Memorial Institute (RPMI) 1640 medium for 2 h at 37°C. The digested supernatant was filtered through a 70 μm cell strainer to obtain a single-cell suspension. Skin lymphocytes were isolated by centrifugation in lymphocyte separation solution (Dakewe Biotech, Beijing, China). CD11c-positive dendritic cells were enriched with the EasySep Mouse CD11c Positive Selection Kit (Stemcell, Cat# 18780) for subtype sorting.

Skin and spleen lymphocytes were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Cat #R8758, Sigma) supplemented with 10% fetal bovine serum (FBS; HyClone, GE Healthcare, Chicago, Illinois, USA), 10% 100 U/mL penicillin and 100 mg/mL streptomycin, and the cells were maintained at 37°C with 5% CO₂. To sort the pDCs subgroup, first use the B cell positive selection kit to isolate B cells (EasySep™ Mouse CD19 Positive Selection Kit, Catalog #18754). Then, the liquid passing through the column of B cell positive selection was performed by EasySep Mouse CD11c Positive Selection Kit (Stemcell, Cat# 18780) to enrich DCs cells. The CD11c-positive DCs were washed three times with PBS, and then added 10μL fluorophore-conjugated antibody (PerCP/Cy5.5-CD103 (Cat#121416), PE/Cy7-CD11c (Cat#117318), FITC-CD11b (Cat#101206), PE-CD45R (Cat#103208) and APC-CD8α (Cat#100712) purchased from BioLegend). The cells were stained for 30 min at 4°C and washed twice prior to flow cytometric analysis (LSR Fortessa, BD) and to sort (Influx, BD).

CD8⁺ T cells were isolated as described above and labeled with 5 µM CellTrace Violet (CTV) (Thermo Fisher Scientific) according to the manufacturer’s protocol. For in vivo proliferation assays, labeled cells were resuspended in HBSS, and 100 µL of cell suspension was injected via the retro-orbital route into anesthetized mice.
To obtain CD11c⁺ DCs for in vitro proliferation assays, spleens were removed from mice and digested in Liberase TL (Roche) for 30 min, and single cell suspensions were obtained by passing the digested spleens through a 60 µm nylon mesh. Magnetic selection of CD11c⁺ DCs was then performed using EasySep Mouse CD11c positive selection kit (STEMCELL Technologies), and the purified CD11c⁺ DCs were pulsed with 1 µg/mL OVA_257–264 at 37°C for 1 h. The DCs were washed three times with fresh medium and cocultured with labeled CD8⁺ T cells at a ratio of 1 DC:10 T cells for 72 h at 37°C, 5% CO₂.
Proliferation index was obtained using FlowJo V10 software.

Spleens of untreated adult mice were digested using Spleen Dissociation Medium (Cat #07915, STEMCELL Technologies). Dendritic cells were isolated by positive selection from the using the EasySep Mouse CD11c Positive Selection Kit (Cat #18758, STEMCELL Technologies). These DC are CD11c positive and more than 90% of them express MHC-II and the costimulatory receptors CD80 and CD86.

DCs were isolated using the EasySep™ mouse CD11c positive selection kit (Stemcell Technologies) following the manufacturer’s protocol. CD4⁺ T cells were isolated by incubating splenocytes with hybridoma supernatants containing mAbs to CD8 (TB105) and MHCII (10.2.16), while CD8⁺ T cells were isolated by incubating splenocytes with hybridoma supernatants containing mAbs to GK1.5 (CD4) and MHCII (10.2.16), all generously provided by the late Charles Janeway Jr. (Yale University), for 30mins at 4°C. Cells were then washed in PBS and incubated for 45mins on ice with magnetic beads conjugated with goat anti-mouse IgG, goat anti-mouse IgM or goat anti-rat IgG (QIAGEN). CD4⁺ T cells were then negatively isolated using magnetic selection. The purity was routinely 90-95%, as verified by flow cytometry.

Bone marrow cells from wild-type B6 or DR4 tg mice were prepared as described above. In a T75 flask, cells were seeded at a density of 1 × 10⁶ cells/mL in RPMI 1640 medium containing 10% heat-inactivated FCS, GlutaMAX-I, 0.1 mM MEM nonessential amino acids, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10 mM HEPES (complete medium), which was further supplemented with 10 ng/mL mouse GM-CSF and IL-4 (PeproTech Inc., Rocky Hill, NJ). Cultures were replenished with fresh medium and cytokines every other day after discarding the floating cells. On day 7, harvested cells were enriched for CD11c⁺ DCs using an EasySep Mouse CD11c Positive Selection Kit (Stemcell Technologies).