Dac00b

The ELISA kit (DAC00B) (R&D system) was used to determine the concentration of activin A in the supernatant of cell cultures as per the manufacturer's instructions.

Peripheral venous blood was collected from healthy and patients’ volunteers using serum separator tubes (10 mL). After 30 min on the benchcoat at room temperature, the tubes were centrifuged at 2000 rpm for 10 min at 4 °C. The collected serum (5 mL) was aliquoted and stored at −80 °C until further use. The concentrations of either GDF8, FOLLISTATIN, GDF11, or ACTIVIN A in the sera were measured using an ELISA kit (respectively # DGDF8, # DFN00, # DY1958, and # DAC00B R&D Systems Europe, Ltd, Abingdon, United Kingdom) according to the manufacturer’s instructions. The optical density was measured using a microplate reader (Infinite 200 Pro, Tecan Group Ltd., Männedorf, Switzerland). Importantly, the myostatin immunoassay was designed to recognize mature GDF8. According to the manufacturer, no significant cross-reactivity or interference was observed in the presence of 50 ng/ml (20 times more than the highest value measured in our experiment) of different proteins including the GDF8 propeptide, GDF11 or GDF15. We have experimentally confirmed this result by using 4000 pg/ml of recombinant GDF11. The absence of cross reactivity with FOLLISTATIN (8000 pg/ml) and ACTIVIN A (4000 pg/ml) was also experimentally validated.

Aliquots of plasma were analyzed for total myostatin, free myostatin (both DGDF80, R&D Systems, UK), FLRG (DFLRG0, R&D Systems, UK), activin A (DAC00B, R&D Systems, UK), FLRG (DFLRG0, R&D Systems, UK), and GDF11 (DY1958, R&D Systems, UK) were analyzed according to manufactures instructions. Total myostatin was measured by the acidification release method. Briefly, 100 μL plasma was treated with 50 μL 1N HCl, vortexed and incubated at room temperature for 10 min then neutralized with 50 μL 1.2 N NaOH and further diluted in 200 μL manufacturer provided diluent for a final dilution of 1:4, similar to previously validated methods (Lakshman et al. 2009). Plasma samples for free myostatin were diluted 1:4 in identical diluent, without acidification or neutralized treatment. All samples and standard were analyzed in triplicate. Of the 88 participants, 56 GDF11 results were either below limit of detection of our assay (31.3 pg/mL) or otherwise undetectable, and are thus not reported here.
As GDF11 has been previously reported to have a high structural similarity to myostatin/GDF8 (Poggioli et al. 2016), we also directly compared the GDF11 ELISA used here against recombinant myostatin in a physiological (0–2000 pg/mL) and supra‐physiological (20,000 pg/mL) range, with no cross reactivity reported (Fig. 1).