Amicon ultra 10k filter devices

In order to test dhCSSCs ability to secrete neurotrophins and analyze their respective effects on neurons, ELISA experiments were performed against the medium in dhCSSCs cultures. Briefly, dhCSSCs were cultivated in 6 well tissue culture plastic plate for 22 days; 1 ml medium aliquots were collected every 3 days from each well. Media samples were then concentrated with Amicon Ultra 10K Filter Devices (Millipore, Merck KGaA, Darmstadt, Germany). Elisa assays for BDNF, GDNF, NT-3, NGF were processed with human BDNF, GDNF, NT-3, NGF Duo set (R&D system, Minneapolis, MN, USA) on n= 3 from two independent experiments.

Western blotting was performed with samples from the cultured medium and the cell lysates (19 (link)). The cell culture media were collected and concentrated using Amicon® Ultra 10K Filter Devices (Millipore). The cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Boston Bio Products, BP-115). Samples were electrophoresed on 8–14% SDS-PAGE and then transferred to Immobilon-P PVDF transfer membranes (Merck Millipore). The membrane was blocked, probed with primary Abs overnight at 4°C, and then incubated with HRP-conjugated secondary Abs (1:3000~5000) at room temperature for 1 h. The protein bands were detected with Odyssey Fc Imager (LI-COR Biosciences). Relative band densities of proteins were quantified using ImageJ Software (NIH).
Immunoprecipitation was performed as described previously (19 (link)). BMDMs were lysed in immunoprecipitation lysis buffer (1% NP-40, 50 mM Tris-HCl, pH 7.4, and 150 mM NaCl) supplemented with 1 mM PMSF and protease inhibitor. The cell lysates were incubated with an appropriate Ab overnight at 4°C, followed by addition of 20 μl protein A/G PLUS-agarose beads for 2 h at 4°C. The resulting immunoprecipitates were dissolved in SDS-PAGE sample buffer for electrophoresis and immunoblot analysis