Human specific enzyme linked immunosorbent assay elisa kits

Total RNA was collected using the Trizol RNA isolation protocol (Thermo Fisher), and 600 ng of total RNA was reverse-transcribed with the QuantiTect Reverse Transcription Kit (Qiagen, Valencia, CA). qPCR was performed using TaqMan1 Universal PCR Master Mix (Thermo Fisher). Primers and probes consisted of p16 (Hs00923894_m1) and RPL13 (Hs00204173_m1) (Thermo Fisher Scientific Life Sciences). qPCR results were normalized to the housekeeping transcript level (RPL13) and the control value of either P5 untreated or P5 CytoD-treated to yield 2^−ΔΔCt,[24 (link)] or the housekeeping transcript level to yield 2^−ΔCt.
Cytokine secretion was analyzed by quantifying the concentration of vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) in MSC spheroid conditioned media 24 h after spheroid formation using human-specific enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN).

We labeled BMP-2 using the Alexa Fluor 488 microscale protein labeling kit (Thermo Fisher) following the manufacturer’s instructions. Fluorescently labeled BMP-2 was adsorbed onto HA and subsequently incorporated into spheroids as described above. Spheroids were imaged using both brightfield and fluorescent microscopy, and these images were overlaid to form a composite image. The relative fluorescence intensity of spheroids in the fluorescent channel was quantified using ImageJ.
In order to quantify morphogen loading, BMP-2 was eluted from the HA nanoparticles as previously reported.^{[25 ]} We prepared an elution buffer composed of 2 mM CaCl₂ solution in N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (HEPES, Thermo Fisher) at pH 6.0. BMP-2-laden HA nanoparticles were incubated in the elution buffer for 40 min, and the supernatant was collected. BMP-2 concentration within the supernatant was quantified using human-specific enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN).