1.5 × 105 CD4+ CD25− T cells and 5.0 × 104 BM pDCs (3:1) were cultured in 96 well U-bottom polystyrene plates (Sigma-Aldrich Co., St Louis, MO) in the presence of recombinant interleukin-2 (IL-2) (final: 10 ng/1ml) (R&D systems, Minneapolis, MN) and platelet-derived tissue growth factor-β (TGF) (final: 10 ng/1ml) (R&D systems, Minneapolis, MN) at 37°C for 4 days. T cell/pDC ratio and time points are considered optimal for induction (unpublished data). To analyze for proliferative changes during culture, T cells were stained with Cell Proliferation Dye eFluor® 670 (eBiosciences, San Diego, CA) before culture at a 5 nM concentration. After 4 days in culture, cells were analyzed for FACS or for in vivo studies by adoptive transfer. All groups in in vitro experiments were done in triplicate. Cell cultures for adoptive transfer were upscaled by a factor of 2 i.e (3.0 × 105 CD4+ CD25− T cells and 1.0 ×105 BM pDCs) in the presence of doubled amount of IL-2 and TGF- β for 4 days incubation.
Recombinant interleukin 2 il 2
Manufactured by R&D Systems
Sourced in United States
Recombinant interleukin-2 (IL-2) is a cytokine that promotes the proliferation and activation of T cells and natural killer cells. It is produced using recombinant DNA technology.
Automatically generated - may contain errors
Lab products found in correlation
Cell proliferation dye efluor 670, by Thermo Fisher Scientific (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
Moflo xdp cell sorter, by Beckman Coulter (1 mentions)
Zoledronate, by Novartis (1 mentions)
Vacutainer cpt cell preparation tube, by BD (1 mentions)
Nk 92 cells, by American Type Culture Collection (1 mentions)
Hek293, by American Type Culture Collection (1 mentions)
Phoenix gp, by American Type Culture Collection (1 mentions)
Mm 1s, by Thermo Fisher Scientific (1 mentions)
Dmem glutamax, by Thermo Fisher Scientific (1 mentions)
Rpmi8226, by American Type Culture Collection (1 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
4 protocols using recombinant interleukin 2 il 2
1
Induction of Regulatory T Cells
2
T Cell Suppression by Plasmacytoid Dendritic Cells
3
Expansion and Transduction of Human γδ T Cells
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Human γδ T cells were expanded and cultured from human PBMCs as described previously.50 In brief, whole blood (7.5–8 mL) was collected in a BD vacutainer CPT cell preparation tube with sodium heparin (BD, Franklin Lakes, NJ, USA) and centrifuged to isolate PBMCs at the interphase. Murine γδ T cells were expanded from splenocytes of C3H mice. Human PBMCs or mouse splenocytes were then cultured in AIM V medium (Thermo Fisher Scientific, Waltham, MA, USA) with recombinant interleukin-2 (IL-2; R&D Systems, Minneapolis, MN, USA) and zoledronate (Aclasta, Novartis, Switzerland) to final concentrations of 1,000 IU/mL and 5 μM, respectively, at the presence of tumor conditioned medium. On day 7, γδ T cells were sorted using a MoFlo XDP cell sorter (Beckman Coulter, Brea, CA, USA).
Sorted γδ T cells were seeded in a 24-well plate and infected with LV-hsa-mir-138 (GENECHEM, Shanghai, China) with 5 μg/mL polybrene, and stable clones were selected and maintained in medium described above with 0.5 μg/mL puromycin.
Sorted γδ T cells were seeded in a 24-well plate and infected with LV-hsa-mir-138 (GENECHEM, Shanghai, China) with 5 μg/mL polybrene, and stable clones were selected and maintained in medium described above with 0.5 μg/mL puromycin.
4
Cell Culture Conditions for Various Cell Lines
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PG13 (ATCC CRL-10686), HEK293 (ATCC CRL-1573) and Phoe-nixGP (ATCC CRL-3215) cells were cultured in Dulbecco's Modified Eagle Medium+ Glutamax (DMEM-Glutamax) with the addition of 10% fetal bovine serum (FBS; Gibco, Billings, MT, USA). K562 (CCL-243), RPMI-8226 (ATCC CCL-155) and MM.1S (CRL-2974) were cultured in Roswell Park Memorial Institute (RPMI) 1640 media (Invitrogen, Carlsbad, CA, USA) enriched with 10% FBS. NK-92 cells (ATCC CSC-C0499) were maintained in Good Manufacturing Practice (GMP)-grade SCGM media (Sartorius CellGenix, Freiburg, Germany) supplemented with 20% FBS and 500 IU/mL recombinant interleukin-2 (IL-2; R&D Systems, Minneapolis, MN, USA) every 2À3 days. Cells were cultured in antibiotic-free conditions and were verified to be mycoplasma free with regular testing. The cells were maintained in a 37°C, 5% CO 2 humidified incubator and split every 2À3 days.
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