Registered: 3 months, 1 week ago
Tal of Larissa.Table 2 Samples informationSample 332OA 393OA 520 524OA 525OA 526OA
Tal of Larissa.Table 2 Samples informationSample 332OA 393OA 520 524OA 525OA 526OA 529OA 530OA 532OA 533OA 150 151 152 153 154 175 Group OA OA OA OA OA OA OA OA OA OA Controls Controls Controls Controls Controls Controls Sex F F M F F F F F F F F M M F M F K-L score 4 4 4 3 4 4 4 4 4 4 1 0 0 0 1Articular cartilage was transported from the surgical room to the laboratory in HBSS medium (Hanks Balanced Salt Solution). Isosulfan blue It was then immediately dissected and subjected to sequential digestion with pronase (1 mg/ml; 90 min; Roche Applied Science, Germany) and collagenase P (1 mg/ml; 3 h; Roche Applied Science, Germany) at 37 . Chondrocytes were cultured in Dulbecco's Modified Eagles Medium/ Ham's F-12 (DMEM/F-12) (GIBCO BRL, UK) plus 5 fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin and were incubated at 37 under a humidified 5 CO2 atmosphere. Chondrocytes were kept in culture for maximum of two passages. Trypsin was used for the detachment of chondrocytes during the primary culture, while type II collagen and type I collagen ratio was screened in all samples to exclude dedifferentiation events.Protein extraction from chondrocytesOA and normal chondrocytes were trypsinized, collected and centrifuged for 10 min at 543xg. The cell pellet was washed with PBS and then centrifuged again for 10 min at 543xg at 4 . The cell pellet was resuspended in RIPA lysis buffer (50 mM Tris/HCl pH 7.2, 150 mM NaCl, 1 Triton X-100, 1 sodium deoxycholate, 0.1 SDS) and incubated on ice for 30 min. Remaining cells where lysed after sonication (3 rounds at 30 amplitute), using a tip sonicator (VCX130, Sonics Materials, Newtown, USA). Samples were clarified via centrifugation (15 min; 13,000xg; 4 ), in a bench top centrifuge, and total protein content was calculated using the BCA method .1D-SDS-PAGE and in-gel digestion10 OA and 6 control chondrocyte samples were studied. OA stage was measured in Kellgren Lawrence system (K-L).Proteins from each sample (50 g) were precipitated using trichloroacetic acid/acetone (final concentration of 25 TCA w/v; 4 ; 30 min). Proteins were precipitated after centrifugation (15,000 g; 30 min; 4 ), in a bench-top centrifuge. Protein pellets were PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/8961164 washed twice with ice-cold acetone, and precipitated via centrifugation, as previously. Excess acetone was aspirated and the protein pellet was air-dried. Precipitated proteins were re-solubilized in a solution containing 1.5 M Tris/HCl pH 8.8 and 6x sample buffer in a ratio of 2:1 and analyzed by 1D-SDS-PAGE 4-10 (29:1 acrylamide/bisacrylamide). Gels were stained with colloidal coomassie blue (Coomassie G250, 10 phosphoric acid, 10 ammonium sulfate, 20 methanol). Each lane was cut in 10 slices, and de-stained after three washes with 50 acetonitrile/water and 50 mM ammonium bicarbonate. Samples were reduced in the presence of 10 mM DTT (45 min; 56 ) and then washed and PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/24059235 alkylated in the presence of 55 mM IAA (45 min; 22oC, shaking, in the dark). Gel slices were washed with 50 mM ABS and proteins were digested with 0.1 g trypsinTsolis et al. Clinical Proteomics (2015) 12:Page 12 of(trypsin Gold, Promega, Fitchburg, Wisconsin), overnight. Generated peptides were collected, after repeated washes with nanopure-H2O, 50 ACN in low binding tubes (Axygen, Union City, CA) and trypsin was quenched by acidifying the sample with 1? L trifluoroacetic acid (TFA), until pH < 2. Peptides were dried under vacuum, during centrifugation (Speedvac; Savant) and desalted using STAG.
Topics Started: 0
Replies Created: 0
Forum Role: Participant