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Lysates and conditioned media were frozen at −70°C until analysis.
Conditioned media were frozen in 1 ml aliquots and stored at -20°C.
After incubation, media were frozen and stored at −20 °C until analysis by UPLC TOFMS and a yeast androgen bioassay.
Homogenates not used to inoculate culture media were frozen at -80°C until used in PCR assays.
At their incubation end points, the various conditioned media were frozen at −30°C and later assessed for their effects on in vitro angiogenesis.
In parallel, conditioned media were collected, cells were separated by centrifugation, proteinase inhibitors were added, and media were frozen prior to analysis for secreted TGF β1 by ELISA (R&D Systems, Minneapolis, MN).
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Conditioned media were generated by culturing E14 progenitors at 50,000 cells/ml for 1 week, after which cultures were centrifuged, media removed and filtered to eliminate any remaining cells or debris and the media was frozen in single use aliquots.
Briefly, the collected superfusion media were freeze-dried and re-dissolved in water.
The fungi were then scrapped off the surface of the media, and the remaining media were freeze-dried.
Total extracellular proteins in the filtrates of cultures grown for 21 days in the above media were freeze-dried, resuspended in 10 mM tartrate (pH 5), impurities removed by a short PAGE run, and stained by Colloidal Blue Kit (Invitrogen, Thermo Fisher Scientific, Waltham, USA).
The pod-based media was freeze-dried and ground into a fine powder.
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