Substrate reagent pack dy999

Poly (D, L-lactide-co-glycolide) (PLGA) 50:50 (12,000 g/mol) was purchased from Evonik Industries (Essen, Germany). Dexamethasone was supplied by Sigma-Aldrich (St. Louis Mo., USA) (purity > 98%). Fibronectin, fibronectin ELISA and reactants (Reagent Diluent DY995, Wash Buffer WA126, Stop Solution DY994 and Substrate Reagent Pack DY999) were purchased from R&D Systems (Minneapolis, MN, USA). Polyvinyl alcohol (PVA, 67,000 g/mol) was obtained from Merck KGaA (Darmstadt, Germany), and methylene chloride was obtained from PanReac AppliChem (Barcelona, Spain).

EGF binding and internalization assays were run in parallel and measured by a non-radioactive method [77 (link)], based on biotin-EGF (Invitrogen). Colon TAFs were plated in two 96-wells plates (20000/well); after 48h cells were switched to SFM for 24h and successively incubated for additional 24h in the presence/absence of Celecoxib. At the end of incubation TAFs were washed twice with cold PBS (with Ca⁺⁺ and Mg⁺⁺) and incubated with 50ng/ml biotin-EGF in SFM (4°C, 45min for binding and 37°C, 30min for internalization). The plate used for the internalization test was then incubated in acidic buffer, pH 3, to eliminate residual membrane-bound EGF. After two consecutive washings with cold PBS, TAFs were fixed and permeabilized. Residual binding sites were blocked by two consecutive incubations with glycine 50mM in PBS and Gelatin 2% + Tween20 0.05% in PBS. Biotin EGF was revealed by incubation with streptavidin-HRP (Life Technologies) 1:15.000 dilution. After extensive washings, TAFs were incubated with the Substrate Reagent Pack DY999 (R&D) and then blocked with DY994 stop solution. Gemini VersaMax spectrophotometer was used to quantify the staining at 450nm. Each experimental point was run in six replicates and data were normalized against controls processed in parallel, either in the absence of TAFs, or with TAFs without biotin-EGF incubation.

ELISA of homogenized whole adipose tissue and supernatants was performed with MAB289 capture (R&D Systems, Abingdon, United Kingdom) and BAF289 detection antibodies (R&D Systems) as a sandwich-ELISA. The tissue used for our studies was not further manipulated, digested with collagenase, or cultured and therefore represents the native state.
Each well was coated overnight with primary antibody solution at a final concentration of 2 μg/ml. Plates were blocked with PBS containing 1% BSA and 5% sucrose for one hour. After incubating samples for another two hours, secondary antibody was added at a final concentration of 200 ng/ml. Streptavidin-POD enzyme conjugate (R&D Systems, Abingdon, United Kingdom, 1 μl/10 ml TBS) was added and left for 20 minutes. Color reaction was determined by the Substrate Reagent Pack DY999 by R&D Systems (R&D Systems, Abingdon, United Kingdom). The reaction was terminated after 20 minutes by adding 0,5 M H₂SO₄ in ddH₂O and plates were read at 450 nm (FLUOstar OPTIMA microplate reader). Bacterially expressed MIF was used as a standard. All assays were carried out in duplicates.