Nylon wool column

HLA-A2 transgenic splenocytes were collected and treated with lysis buffer to eliminate red blood cells, washed, and re-suspended in RPMI-1640 (Giboco BRL) with 10% FBS (Giboco BRL). Lymphocytes were derived from splenocytes using nylon wool columns (Wako, Japan). Single-cell suspensions of lymphocytes (2 × 10⁶ cells/well) were grown in six-well plates (Corning). The purities of the isolated T cells were determined by flow cytometry analysis after staining with anti-CD3- PE-Cy5 (eBioscience, United States), and the samples with purity of more than 80% were used for this experiment.

PBMCs were separated from heparinised blood with the standard Ficoll-hypaque (GE Healthcare, USA) gradient centrifugation method and washed twice in Ca²⁺/Mg²⁺-free PBS pH 7.4. The PBMCs were resuspended to a final density of 1 × 10⁶ cells/ml in RPMI 1640 (Invitrogen, USA) containing 10% heat inactivated fetal calf serum (FCS), 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen, USA). To obtain monocytes, the PBMCs were plated in 24-well culture plates and incubated for 2 h at 37°C in 5% CO₂. The non-adherent cells were aspirated and incubated separately under the same conditions for later use.
T cells were isolated from the non-adherent population. The non-adherent cells were subject to nylon wool columns (Wako, Japan) as previously described
[32 (link), 33 (link)]. Briefly, the columns were washed and equilibrated with warm (37°C) complete RPMI. 3 × 10⁸ cells were suspended in 2 ml of warm complete RPMI, added into the column and incubated at 37°C for 60 min. The column was eluted with 20 ml of warm complete RPMI and the T cells were recollected in a tube. Viability of monocytes and T cells was > 95% in all the experiments as measured by trypan blue exclusion test.

T lymphocytes were obtained from splenocytes with nylon wool columns (Wako) in accordance with a previously described protocol (20 (link)). P815/c cells (5 × 10⁴ cells/well) seeded in 96-well plates and incubated with antigen for 24 h were used as target cells. T lymphocytes, used as effector cells, were cultured with P815/c cells at a specific E:T ratio (10:1) at 37°C for 4 h. The Cytotoxicity LDH assay kit (Dojindo) was used to assess LDH. The optical density values of the supernatants were recorded at 490 nm. Cytotoxicity (%) was calculated as follows: ([experimental release – effector spontaneous release – target spontaneous release]/[target maximum release – target spontaneous release]) ×100%. Flow cytometry was used to classify T lymphocytes, by staining with PerCP-Cy5.5-conjugated anti-CD8, PE-conjugated anti-CD4, PE-Cy7-conjugated anti-IL-4, and APC-conjugated anti-IFN-γ antibodies.

Splenic T cells were isolated from female Foxp3/GFP knock-in C57BL/6 mice using a nylon-wool column (Wako, Osaka, Japan) and stained with Violet Proliferation Dye 450 (Becton Dickinson) then used as responders. Two types of MLR experiments were performed. In the first experiment, the generated iPS-DCregs, BM-DCregs and BM-DCcons were used as stimulator cells, and all cells were studied at three amounts (1 × 10⁴, 2 × 10⁴ and 4 × 10⁴) and co-cultured with responder T cells (2 × 10⁵) in the wells of 96-well round-bottomed culture plates (Greiner bio-one) for four days.
In the other experiment, matured BM-DCcons were used as stimulator cells (1 × 10⁴) and treated with X-ray irradiation (20 Gy) and co-cultured with responder T cells (2 × 10⁵) and the regulators (iPS-DCregs, BM-DCregs or BM-DCcons), and all cells were studied at three amounts (1 × 10⁴, 2 × 10⁴ and 4 × 10⁴) in the wells of 96-well round-bottomed culture plates (Greiner bio-one) for four days. At the end of the culture, the cells were harvested and stained with PE-Cy7-CD8 and APC-CD4. The stained cells were analyzed using FCM.