Cd4 cd25 regulatory t cell isolation kit

Aβ-specific CD4⁺CD25^- effector T cells (Aβ-Teffs) were isolated from the splenocytes of Aβ-immunized DEREG mice using a CD4⁺CD25⁺ Regulatory T Cell Isolation Kit (Miltenyi Biotec) and labeled with Cell Proliferation Dye eFluor 670 (e670; Invitrogen, #65-0840-85) according to the manufacturer's protocol. Labeled Aβ-Teffs were seeded at 1 × 10⁵ cells per well in round-bottom 96-well plates coated with 5 µg/mL anti-CD3e antibodies and containing 2 µg/mL soluble anti-CD28 antibodies and cocultured with Aβ⁺, KLH⁺ or PBS⁺ Tregs at a ratio of 1:1, 1:2 or 1:4 (Treg:Teff). After 3 days, Teff proliferation was analyzed as previously described 7 (link). % Suppression was calculated as 100 - (proliferation [Treg coculture]/proliferation [Teff only]) × 100.

Splenocytes from DEREG mice used to generate PBS⁺ Tregs, Aβ⁺ Tregs or KLH⁺ Tregs were obtained by mechanical disruption of spleen. The splenocytes were passed through a 40-μm cell strainer and cultured with 5 µg/mL plate-bound anti-CD3ε antibodies in combination with 2 µg/mL soluble anti-CD28 antibodies (BD Biosciences). The cells were stimulated with Aβ (10 µg/mL) or KLH (10 µg/mL) and bvPLA2 (0.4 µg/mL). After 4 days, CD4⁺CD25⁺ Tregs were isolated using magnetic-activated cell sorting (MACS) according to the manufacturer's protocol (CD4⁺CD25⁺ Regulatory T Cell Isolation Kit, Miltenyi Biotec).

Tregs were isolated from mesenteric, axillary, inguinal, and cervical lymph nodes using the CD4⁺CD25⁺ Regulatory T Cell Isolation Kit (Miltenyi Biotec), using a two-step procedure. First, non-CD4⁺ cells were magnetically labeled with a cocktail of biotin-conjugated antibodies against CD8, CD11, CD45R, CD49b, Ter-119, and Anti-Biotin MicroBeads. The labeled cells were subsequently depleted using a MACS LS column (Miltenyi Biotec). In the second step, the flow-through fraction of pre-enriched CD4⁺ T cells was labeled with CD25 MicroBeads for subsequent positive selection of CD4⁺CD25⁺ Tregs. Isolated cells were counted and seeded into 96-well round bottom plates, previously activated overnight at 4 °C with an anti-mouse CD3 antibody in PBS (1:1000, Invitrogen) and cultured in RPMI-1640 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 10% FBS, and recombinant murine IL2 (50 ng/ml). The supernatants produced by Treg cells were recovered at 24–48–72 h and stored at −80 °C.

Spleens and lymph nodes (popliteal/ inguinal) were collected from mice 3 days post-arthritis induction and dissociated as described. Splenocytes and lymph node cells were seeded separately at 1.0 × 10⁶ cells/well in 96-well plates (Sarstedt) in RPMI-1640 with 10% FBS, 0.05 μg/mL IL-2 and 10 μg/ml brefeldin A (Sigma) and activated with cell stimulation cocktail (eBioscience) at 37 °C for 4 hours. No cell stimulation cocktail and no brefeldin A served as controls. Following activation, cells were resuspended in 2 mM EDTA in PBS and Tregs (CD4+ CD25+ FOXP3+) were isolated using the CD4+ CD25+ Regulatory T Cell Isolation Kit (Miltenyi) following manufacturer’s instructions; or stained for T cell subset identification. For this, cells were permeabilised using permeabilisation buffer kit (eBioscience) and intracellularly stained with anti-mouse IFNγ (Th1), IL-4 (Th2) or IL17a (Th17) (eBioscience). Cells were analysed on a BD FACS Canto II flow cytometer and comparisons drawn for percentage CD4+ cells and signal intensity (XGeoMean) for each antibody.

Spleens from treatment and control animals sacrificed at 2 (n = 4) and 3 (n = 6) days post-arthritis induction were weighed and dissociated in PBS using a 7 ml borosilicate glass dounce tissue grinder with loose pestle (Fisherbrand). Cells were passed through a 30μm pre-separation MACS SmartStrainer (Miltenyi), centrifuged and resuspended in PBS pH 7.2 containing 0.5% bovine serum albumin and 2 mM EDTA. Tregs were isolated using CD4+ CD25+ Regulatory T Cell Isolation Kit (Miltenyi) following manufacturer’s instructions. CD4+ CD25+ cells were stained with CD4-FITC, CD25-APC and FOXP3-PE and analysed on a BD FACS CANTO II (BD Biosciences).

Splenocytes were isolated as described before. CD4⁺CD25^– Tconv and CD4⁺CD25⁺ Treg were isolated as described above using the CD4⁺CD25⁺ Regulatory T cell Isolation Kit (MACS, Miltenyi Biotec). Tconv were stained with CFSE to track cell proliferation and co-cultured with Treg (4:1 ratio, stimulation with 0.5 µg/mL plate-bound anti-CD3, 1 µg/mL soluble anti-CD28). Cells were cultured for 3 days (37 °C, 5% CO₂) prior to FACS analysis. For antigen-specific suppression assays, Treg from the respective mouse line and Tconv from 2D2 mice were isolated and labeled with CFSE as described before. For isolation of APCs from WT mice, the spleen was homogenized by enzymatic digestion. Therefore, 0.1 mg/mL collagenase D was injected into the spleen and incubated for 15 min at 37 °C. Spleen was homogenized by a 70 µm cell strainer. To isolate CD11c⁺ APCs, CD11c MicroBeads (MACS, Miltenyi Biotec) were used in a MACS separation according to the manufacturer’s protocol. APCs were incubated with 20 µg/mL MOG_35–55 for 10 min at 37 °C and washed with PBS before setting up the culture. A co-culture of 5 × 10⁴ APCs and in total 2 × 10⁵ Tconv and Treg (ratios of 1:1, 2:1 and 4:1) was set up for 3 days. Flow cytometry analysis of CFSE-labeled Tconv was used to determine the suppressive capacity of Treg.

As previously described [12 (link)], CD4⁺CD25⁻T cells were purified from the spleens of D1 mice using a CD4⁺CD25⁺ regulatory T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and were subsequently stimulated with anti-CD3/CD28 monoclonal antibodies (mAbs, 50 ng/mL, PeproTech) in the presence of TGF-β1 (5 ng/mL, PeproTech) for 5 days. The T cells were incubated with anti-mouse CD4-FITC mAb, fixed, permeabilized, and stained with anti-mouse FoxP3-PE or isotype control mAbs (BD Biosciences Pharmingen). And the suppressive activity of these cells against T cell proliferation was examined using an in vitro suppressive assay. Approximately 3 × 10⁶ cells were transferred to CIA mice at 25 days after the first CII immunization.