Dynabeads untouched mouse cd8 cells kit

Splenocytes were isolated from wild-type (WT) B6 mice, CD8⁺ T cells were isolated by using the Dynabeads Untouched Mouse CD8 Cells Kit (Thermo Fisher Scientific), seeded in 96-well round-bottom culture plates (3 × 10⁵ cells/well), and cultured with 2 µg/mL soluble anti-CD3e mAb (clone 145-2C11, BioLegend) and 1 µg/mL soluble CD28 (clone 37.51, BioLegend) for 24 h. Stimulated CD8⁺ T cells were analyzed using an LSR II or Fortessa flow cytometer (BD Biosciences).

CD8⁺ T cells were negatively isolated from murine splenocytes by using the Dynabeads™ Untouched™ Mouse CD8 Cells Kit (ThermoFisher). Murine CD8⁺ T cells were cultured in AIM V medium, supplemented with 10 % FCS, 100 U/ml IL-2, and 50 μM β-Mercaptoethanol, without stimulus. For the stimulation, the above mentioned medium was supplemented with Dynabeads™ Mouse T-Activator CD3/CD28 for T-Cell Expansion and Activation (ThermoFisher). The stimulated CD8⁺ T cells were kept in the medium with a 5:4 cell-to-bead ratio at 37 °C and 5 % CO₂ for up to 4 days.

C57BL6/J mice were purchased from Charles River Laboratories and bred in our own colonies. All animal experiments were approved by local authorities and were performed in compliance with the German Animal Protection Law (Tierschutzgesetz, §11, Abs.1 Nr.1). Mice were housed under specific‐pathogen‐free conditions and sacrificed by cervical dislocation at the designated time. Only female mice between 12 and 24 weeks (adult mice) and 78 and 102 weeks (elderly mice) were used for this study. Mice with splenomegaly (spleen to body weight ratio above 0.6) or macroscopically visible tumors were excluded.
Spleens were harvested and splenocytes were isolated using a 40 μm cell strainer (Corning®). Erythrocytes were lysed by incubation with a hypoosmolar solution. CD8⁺ T cells were negatively isolated using the Dynabeads™ Untouched™ Mouse CD8 Cells Kit (ThermoFisher). Naïve CD8⁺ T cells were isolated with the Naive CD8a⁺ T Cell Isolation Kit (Miltenyi). The purity and subtype distribution were evaluated by flow cytometry.

Male C57BL/6J mice were injected with streptozotocin (STZ) intraperitoneally using a single high dose injection protocol (27 (link)). STZ was administered in a concentration of 150 mg/kg body weight diluted in a 0.1 M sodium citrate buffer. Blood samples were taken from the tail vein and glucose levels were tested by standard test strips (Accu-Chek) twice a week. Additionally, overall health status and body weight was monitored and evaluated using a score sheet over the whole observation period. One week after injection, the blood sugar levels were evaluated. Mice with a blood glucose level of more than 280 mg/dL one week after the injection were considered as diabetic and were sacrificed at day 29 after injection. Spleens were removed from mice and kept in sterile PBS on ice until isolation. Splenocytes were isolated with a 40 µm cell strainer (Corning) and erythrocytes were lysed using a hypo-osmolar solution. CD8⁺ T cells were isolated with the Dynabeads™ Untouched™ Mouse CD8 Cells Kit (ThermoFisher Scientific). Purity of isolated cells was evaluated by flow cytometry. Isolated CD8⁺ T cells were stimulated with Dynabeads Mouse T-Activator CD3/CD28 (ThermoFischer Scientific) for 3 days and cultured in AIM V medium (ThermoFisher Scientific) containing 10% FCS, 50 µM ß-Mercaptoethanol and 100 U/ml recombinant human IL-2 (Miltenyi Biotec).

CD8⁺ T cells were isolated from the spleens of WT B6 mice and Batf^−/−Batf3^−/− mice using the Dynabeads Untouched Mouse CD8 Cells Kit (Thermo Fisher Scientific). The purified CD8⁺ T cells reached over 95% (Additional file 1: Figure S1). Half million CD8⁺ T cells were injected into Rag1^−/− mice intravenously (i.v.) on day − 1 after injection. BALB/c skin were transplanted into Rag1^−/− mice on day 0.