PAF-stimulated Th17 development is dependent on cytokines, LC-T cell contact and selected signaling pathways.
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(a) Monocyte-derived LC were stimulated with either vehicle or PAF in the presence of neutralizing Ab to IL-15, IL-6R, and/or IL-23, or control Ig, and cocultured with antiCD3/CD28-activated CD4+ T cells for 5 days.
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Cell Culture
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Frozen female CD34+ CBCs were supplied by Bio-Resource Center .
- C34+ CBCs were cultured in hematopoietic culture medium [serum-free X-Vivo10 containing 50 ng/mL IL-6 , 50 ng/mL sIL-6 , 50 ng/mL SCF , ten ng/mL TPO , and 20 ng/mL Flt3/4 ligand ].
- Reprogrammed cells were cultured in feeder-less primate ES cell medium Repro FF (, .
- No. RCHEMD004), ReproFF2 (ReproCELL, cat No. RCHEMD006), mTeSR1 ( catalog number 05850) or E8 (16) supplemented with five ng/mL bFGF (total bFGF ten ng/mL) on Pronectin F-coated dishes.
- Passage of human iPSCs was previously described
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Cell proliferation assay
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HEK293T cells were transfected with mc_mock, mc_sTNFR-Fc, or mc_anti-IL-6R and the conditioned media were collected 24 h post-transfection.
- A-FLS were incubated with or without human IL-6 (100 ng/mL& , , ), sIL-6 (100 ng/mLocky , ), and TNFα (20 ng/mL& ) for 72 h. uring the incubationA-FLS were treated with the conditioned media of HEK293T cells transfected with mc_mock, mc_sTNF-Fc, mc_anti-IL-6 or PBS (as a negative control).
- Cell proliferation was assessed using the cell counting kit-8 (CCK-8 , , ), according to the manufacturer's instructions.
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Enzyme-linked immunosorbent assay (ELISA)
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To analyze the amounts of anti-IL-6R antibody and sTNFR2-Fc protein expressed by the transfected cells, the culture media of HEK293T cells transfected with the indicated minicircle vectors were analyzed by ELISA at 24 h post-transfection.
- The levels of sTNFR2-Fc were quantified using human sTNF-R (80 kDa) Platinum ELISA (eBioscience, San Diego, CA), according to the manufacturer's instructions.
- ELISA was performed to determine the levels of anti-IL-6R antibodies.
- Briefly, a 96-well microtiter plate was coated with commercial anti-human IL-6R antibodies (B-R6; eBioscience) at a concentration of 0.5 μg/mL in coating buffer, and incubated overnight at 4°C.
- The plate was washed five times with washing buffer, and incubated with 1 μg/mL of human sIL-6R at room temperature (RT) for 1 h. After washing, the plate was incubated with blocking solution for 1 h at RT, followed by incubation with 100 μL of serially diluted tocilizumab (used as a standard), and the conditioned media from HEK293T cells were transfected…
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To analyze the amounts of anti-IL-6R antibody and sTNFR2-Fc protein expressed by the transfected cells, the culture media of HEK293T cells transfected with the indicated minicircle vectors were analyzed by ELISA at 24 h post-transfection.
- The levels of sTNFR2-Fc were quantified using human sTNF-R (80 kDa) Platinum ELISA (eBioscience, San Diego, CA), according to the manufacturer's instructions.
- ELISA was performed to determine the levels of anti-IL-6R antibodies.
- Briefly, a 96-well microtiter plate was coated with commercial anti-human IL-6R antibodies (B-R6; eBioscience) at a concentration of 0.5 μg/mL in coating buffer, and incubated overnight at 4°C.
- The plate was washed five times with washing buffer, and incubated with 1 μg/mL of human sIL-6R at room temperature (RT) for 1 h. After washing, the plate was incubated with blocking solution for 1 h at RT, followed by incubation with 100 μL of serially diluted tocilizumab (used as a standard), and the conditioned media from HEK293T cells were transfected with minicircle vectors for an additional 2 h. After washing the plate, an anti-hIgG-HRP antibody (AP112P , , ) was applied to the wells and incubated for 1 h at RT.
- The plate was washed seven times and then incubated with the TMB substrate solution (eBioscience) for 15 min.
- After applying stop solution, the absorbance was measured at 405 nm.
- At 10 days after the injection of minicircles, 100 μL of venous blood was collected from the orbital sinus of anesthetized mice.
- The levels of anti-IL-6R antibodies and sTNFR2-Fc in serum were analyzed as described above.
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