Trypsin

GFP-22 siRNA was purchased from Qiagen (USA) and scrambled siRNA (custom 22 mer) was purchased from GE Healthcare Dharmacon (USA) (Sense Strand: 5’- GCA AGC TGA CCC TGA AGT TC (dTdT)-3’, anti-sense strand 3’- GAA CUU CAG GGU CAG CUU GC-5’, Mol. Wt. 13,965.6 (g/mol), the oligonucleotide has been converted to the 2’-hydroxyl, and annealed), Inc. MTS reagent was purchased from Promega (USA). Lipofectamine^® 2000 (L2K) transfection reagent, SYTOX^® Red dead cell stain, LysoTracker^® Blue DND-22, and AlexaFluor^®680 Wheat Germ Agglutinin- (AlexaFluor^® 680 WGA) conjugate were purchased from Life Technologies (USA). Label IT^® siRNA Tracker intracellular localization kit (Cy3) was obtained from Mirus (USA). Fetal Bovine Serum (FBS), DMEM and RPMI media, sodium pyruvate, PBS and trypsin, were purchased from Atlanta Biologicals (USA). Mark-tubes made of borosilicate glass #50, (L= 80 OD= 1.50 Wall= 0.01 mm) were purchased from Hilgenberg GmbH (Germany).

MDCK cell strain-2 obtained from ATCC (Rockville, MD, USA) was grown in 75 cm² Corning polystyrene flasks (Corning, NY, USA). Dulbecco’s modification of Eagle’s medium (Cellgro, Manassas, VA, USA), which was enriched with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, VA, USA), 100 units/mL penicillin, 100 μg/mL streptomycin (Atlanta Biologicals), and 1.42 g/L HEPES sodium was used to culture the cells. The cells were incubated at 37°C, 95% humidity, and 5% CO₂. The medium was changed three times a week, and the cells were trypsinized with 0.25% trypsin in 1.0 mM EDTA (Atlanta Biologicals). One-tenth of the harvest cells were seeded in a new polystyrene flask. The MDCK cells used were between passage number 45 and 65.

Articular chondrocytes were isolated according to a published procedure
[36 (link)]. Briefly, untreated C57BL/6J
mice were sacrificed and articular cartilages from both knee joints were
collected, rinsed in PBS, and incubated with 2.5% trypsin (Atlanta Biologicals,
Flowery Branch, GA) in 3 mL of PBS at 37°C for an hour. The trypsin was
removed and the tissue was digested with Pronase (2 mg/mL) in Minimal Essential
Medium α Medium (α-MEM) containing penicillin and streptomycin at
37 °C for 1 hr. The Pronase was removed and the exposed articular
cartilage layer was then digested overnight with 0.25 mg/mL of collagenase IV in
α-MEM containing 5% FBS and antibiotics. The digestion was terminated by
the addition of α-MEM containing 10% FBS. The released articular
chondrocytes were filtered through a 70-μ sterile filter, collected by
centrifugation, and expanded in α-MEM supplemented with 10% FBS. The
isolated cells expressed predominantly type II collagen (a marker for
proliferative chondrocytes) with 400- to 500-fold lower levels of type X
collagen (a marker for hypertrophic chondrocytes) or type I collagen (a marker
for fibroblasts and osteoblasts) (Fig. 1C),
confirming that these are bona fide chondrocytes. These cells also expressed
substantial amounts of Sox9 and Rho-associated protein kinase (ROCK), two of the
genes that are essential for cartilage matrix production [37 (link)].