Dynabeads cd25

Extraction of CD25 positive cells was subsequently performed after CD4 isolation using Dynabeads CD25 and DETACHaBEAD CD4/CD8 (Invitrogen). After isolation, 2 × 10⁴ cells were cultured in 96 round-bottom well plate in culture medium (RPMI 1640 medium supplemented with 10% FBS, 2 mm Glutamax, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin (Invitrogen). Dynabeads Human T_reg Expander (Invitrogen) was added at a beads-to-cell ratio of 2:1. This was called a standard method. The expander was a mixture of anti-CD3 and anti-CD28 antibodies plus IL2. Also some expansion protocols also had added rapamycin (1 μM, EMD Biosciences, San Diego, CA, USA) plus TNF (20 ng ml⁻¹), or plus TNFR2 agonistic antibody (2.5 μg ml⁻¹, Immunobiology Core, MGH, Boston, MA, USA). After two days, IL-2 (200 U ml⁻¹) was added to the culture. Half of the media was changed every 2–3 days containing rapamycin (until day 7) and 100 U ml⁻¹ of IL-2. On day 9, additional TNF or TNFR2 mAbs were supplied into the media. On day 16, cells were collected, Dynabeads Human T_reg Expander was removed, washed and rested at 37 °C in a humidified 5% CO₂ incubator in RPMI 1640 medium supplemented with 1% FBS, 2 mm Glutamax, 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin and 10 U ml⁻¹ IL-2. On the following day, cells were counted using hemacytometer, and their phenotype was analyzed by flow cytometer.

The coding sequence of HIRIP3 was PCR-amplified and sub-cloned into the XhoI-NotI sites of the pREV-HTF retroviral vector in frame with an N-terminal FLAG and HA tags. Retroviruses were produced in Phoenix retrovirus packaging cells and used to infect target HeLa cells, as described previously [24 (link)]. Briefly, Phoenix cells were transfected using pREV-HTF and viral particles were used to transduce HeLa cells. Stably transduced HeLa cell populations were sorted by triple immunomagnetic selection using Dynabeads cd25 (Invitrogen, Waltham, MA, USA). Positive cells were checked by immunofluorescence using standard procedures [25 (link)]. Rat anti-HA antibody (Roche, Basel, Switzerland) was used at 1/200 dilution; the secondary antibody goat anti-Rat IgG coupled to Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA) was used at 1/400 dilution. Selected cell line expressing HIRIP3 protein fused to N-terminal FLAG- and HA-epitope tags (e-HIRIP3) was grown in suspension culture to generate four liters. Cells were maintained in incubator equipped with HEPA (high-efficiency particulate arrestance) filter, at 37 °C and 5% CO₂. Cells were grown in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% FBS and 0.1 mg/mL penicillin/streptomycin solution. Cell pellets were collected by low-speed centrifugation and stored at −80 °C.

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized peripheral blood using Ficoll–Paque gradient centrifugation (Sigma-Aldrich, St. Louis, MO, USA). CD19⁺ B cells were isolated by magnetic bead-based positive selection using Dynabeads and DETACHaBEAD CD19 (Invitrogen). CD4⁺ T cells were isolated with Dynabeads CD4 (invitrogen) and CD25⁺ T cells were depleted from the CD4⁺ T cell population using Dynabeads CD25 (invitrogen). All isolations were done according to the manufacturer’s instructions. The purity of isolated CD19⁺ B cells was consistently >95% as analyzed by flow cytometry.