Recombinant human fgf basic 154 a a

Transfected HEK293T cells were washed with DMEM containing 1% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin for 18 h, and incubated with the concentrations indicated in the respective figures for 36 h (unless stated otherwise) with one of the following reagents: recombinant human VEGF165 (cat. no. 100-20, Peprotech, London, UK), recombinant human FGF-basic (154 a.a.) (cat. no. 100-18B, Peprotech, London, UK), recombinant human TNFα (cat. no. 300-01A, Peprotech, London, UK), or recombinant human TGF-β1 (cat. no. 100-21, Peprotech, London, UK). Transfected hMSC-TERT cells were stimulated in DMEM containing 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. Jurkat cells were centrifuged and resuspended in fresh growth medium containing the indicated concentrations of ionomycin (cat. no. I0634-1MG, Sigma-Aldrich) and phorbol 12-myristate 13-acetate (PMA; cat. no. P1585-1MG, Sigma-Aldrich).

Rosette model is also based on a published method (Shi et al., 2012a (link); Shi et al., 2012b (link)). When the H9 cells in the 6-well plate grow to ∼95% confluence, the mTeSR™1 medium was replaced with the filter-sterilized 3NM medium, which consisted of 40 ml N2B27 medium, .4 μmol SB-431542 (MCE), .04 μmol Dorsomorphin (MCE), and 100 μg Insulin (human) (MCE). The medium was changed every day. After 8–10 days (when an obvious thickened and whitened culture layer can be observed with the naked eyes), the cells were passaged. Cells from one well of the 6-well plate were equally divided into 24 wells of a 48-well plate. 9 mm cell slides (required for confocal microscopy imaging) were placed in the 48-well plate before cells are seeded, and wells were coated with Matrigel^®, which is diluted as in the embryonic stem cell culture. One day after passage, the medium was changed to N2B27 medium supplemented with 20 ng/ml Recombinant Human FGF-basic (154 a.a.) (PEPROTECH), and the medium was changed every day. The fourth day after passage was the endpoint of the rosette culture.

The following GSC culture medium was applied to enrich GSCs: DMEM/F12 medium (Thermo Fisher Scientific, Grand Island, NY) supplemented with 1 × B-27™ Supplement, serum free (Gibco, 17504044), 20 ng/ml Animal-Free Recombinant Human EGF (PeproTech, AF-100-15), 20 ng/ml Recombinant Human FGF-basic (154 a.a.) (PeproTech, 100-18B), and 1× penicillin–streptomycin. Cells were seeded in Costar^® 24 Well Clear Flat Bottom Ultra Low Attachment plates (Corning, NY) at 1000 cells/ml. Spheres were carefully aspired into 15 ml tube, and spin down at 1000 rpm for 3 min. Subsequently, 1 ml warm trypsin was used to digest spheres at room temperature for 3 min. The reaction was stopped by adding 10 ml GSC culture media, then single cells were spin down and re-plated.