Quantifying Serum and Vertebral TGFβ1 Levels

Serum levels of osteocalcin and TRACP5b were tested by ELISA according to the manufacturer's instructions^{12 ,15 (link),29 (link)}. Levels of TGFβ1 in mouse serum were measured using a mouse TGFβ1 DuoSet ELISA kit (#DY1679) and in human vertebral specimens using a human TGFβ1 Quantikine ELISA Kit (#DB100B) from R&D Systems according to the manufacturer's guidelines. Briefly, total TGFβ1 levels were measured in 40 μl of serum or 10 μg of vertebral protein lysates by mixing these with 20 μl 1 N HCl, followed by neutralization with 20 μl 1.2 N NaOH/0.5 M HEPES, according to the manufacturer’s sample activation procedure; levels of endogenously active TGF-β1 in these samples were measured by skipping this sample activation procedure. These samples then were diluted 1:20 in assay diluent for measurement of total TGFβ1, or they were diluted 1:4 in assay diluent for measurement of active TGFβ1. ELISA plates were analyzed by investigators blinded to sample identity by reading absorbance using a microplate reader set to 450 nm with wavelength correction set to 540 nm.

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Li J., Ayoub A., Xiu Y., Yin X., Sanders J.O., Mesfin A., Xing L., Yao Z, & Boyce B.F. (2019). TGFβ-induced degradation of TRAF3 in mesenchymal progenitor cells causes age-related osteoporosis. Nature Communications, 10, 2795.

Publication 2019

mouse TGFβ1 DuoSet ELISA kit human TGFβ1 Quantikine ELISA Kit

Assay Elisa Hepes Human Mouse Osteocalcin Protein Serum Tgfβ1 Vertebral

Corresponding Organization :

Other organizations : University of Rochester Medical Center

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Serum levels of osteocalcin
Serum levels of TRACP5b
Levels of TGFβ1 in mouse serum
Levels of TGFβ1 in human vertebral specimens

control variables

Measurement of endogenously active TGF-β1 in the samples by skipping the sample activation procedure

controls

Positive control: None mentioned
Negative control: None mentioned

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