Amicon ultra 15 3 kda

Conditioned medium (15 mL) was harvested from MEF and MEF-Has3-KO cells cultured by standard protocols for 24 h. After that, samples with different MW cutoffs were concentrated according to the standard protocol by using the following ultracentrifugal filters: Amicon^® Ultra-15-100 kDa (UFC910008, Merck Millipore, Burlington, MA, USA), Amicon^® Ultra-15-50 kDa (UFC905008, Merck Millipore, Burlington, MA, USA), Amicon^® Ultra-15-30 kDa (UFC903008, Merck Millipore, Burlington, MA, USA), and Amicon^® Ultra-15-3 kDa (UFC900308, Merck Millipore, Burlington, MA, USA). The concentration of HA (ng/mL) in concentrated samples was determined by using an HA ELISA-like assay [14 (link)].

When either hMSCs or mBMSCs (P2 passage in culture) reached 80% of confluence, they were rinsed three times in PBS to remove any residue of FBS. The cells were then replenished with a serum-free (SF) medium (D-MEM for hMSCs or alpha-MEM for mBMSCs) supplemented with 2 mM of L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin, without FBS, for 24 h.
For the preconditioning of the hMSCs, the SF medium was discarded, and the cells were cultured in the SF medium supplemented with inflammatory stimuli (50 ng/mL Tumor Necrosis Factor-α (TNF-α) (PeproTech, London, UK) and 50 ng/mL Interleukin 1-α (IL1-α) (PeproTech, London, UK) under hypoxic condition (1% O₂) (Hyp^INF) for 24 h.
Subsequently, the CM was collected from both cell sources and concentrated with Amicon-Ultra-15, 3 kDa centrifugal filter tubes (Merck, Darmstadt, Germany). In order to quantify the protein amount, a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used. The CM samples were then stored at −80 °C and used in experiments as 50 μg.