Immature murine articular chondrocytes in primary culture: a new tool for investigating cartilage Colette Salvat, B.Sc., Audrey Pigenet, Lydie Humbert,

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Immature murine articular chondrocytes in primary culture: a new tool for investigating cartilage Colette Salvat, B.Sc., Audrey Pigenet, Lydie Humbert, Francis Berenbaum, Ph.D., Sylvie Thirion, Ph.D., M.D. Osteoarthritis and Cartilage Volume 13, Issue 3, Pages 243-249 (March 2005) DOI: 10.1016/j.joca.2004.11.008 Copyright © 2004 OsteoArthritis Research Society International Terms and Conditions

Fig. 1 Sample collection and establishment of primary cultures of iMACs. (A) Skin and soft tissues were removed from the hind legs of newborn mice. (B) The femurs were dislocated and the soft tissue about the joints was discarded. (C) The femoral heads, condyles, and tibial plateaux were isolated. (D) A cartilage piece after overnight incubation with collagenase D. (E–H) Morphology of mouse newborn articular chondrocytes plated on culture dishes. Phase-contrast micrographs of cells in primary cultures after 2 (E), 3 (F), 5 (G), and 6 (H) days (Div: Days in vitro) (Bars=20μm). Osteoarthritis and Cartilage 2005 13, 243-249DOI: (10.1016/j.joca.2004.11.008) Copyright © 2004 OsteoArthritis Research Society International Terms and Conditions

Fig. 2 Morphology and staining of iMACs in primary cultures. (A) Morphology of iMACs seen under a phase-contrast (PC) microscope after fixation. (B) Immunostaining with an anti-type II collagen antibody. (C) Staining with Alcian Blue. (D) Immunostaining with an ACPG antibody (Bars=20μm). Osteoarthritis and Cartilage 2005 13, 243-249DOI: (10.1016/j.joca.2004.11.008) Copyright © 2004 OsteoArthritis Research Society International Terms and Conditions

Fig. 3 iMAC phenotype and loss of chondrocyte differentiation of passaged iMACs. (A) Effect of passages on the production of type II collagen, type I collagen, and aggrecan mRNA by iMACs, as determined using real-time PCR, with GAPDH as the control. (Values are the means±s.e.m., type II collagen: P0, n=16; P1, n=5; P2, n=8; P3, n=5; P4, n=5; aggrecan: P0, n=16; P1, n=8; P2, n=8; P3, n=6; P4, n=6; type I collagen: P0, n=8; P1, n=5; P2, n=6; P3, n=6; P4, n=5; **P<0.01 and ***P<0.001 compared to control, by Student t test). (B) Effect of passages on the production of type II collagen and aggrecan protein as determined by Western blotting (the data represent a typical experiment conducted three times with similar results). Osteoarthritis and Cartilage 2005 13, 243-249DOI: (10.1016/j.joca.2004.11.008) Copyright © 2004 OsteoArthritis Research Society International Terms and Conditions

Fig. 4 Functional characterization of primary cultured murine articular chondrocytes. iMACs were incubated with 10ng/ml IL-1β for 24h and (A) PGE2 release was measured without (n=7 for control and IL-1β alone) or with (n=4) 10−7M dexamethasone. (B) Type II collagen and COX-2 protein levels were evaluated by immunoblot (representative image of three independent experiments). (C) NO2 release was detected in cell supernatants with vehicle, 10−7M dexamethasone (n=7), or 5.10−4M DETC (n=6) prior to IL-1β stimulation (n=11 for control and IL-1β alone). Asterisks denote a statistically significant difference from control values. *P<0.001. # denotes a statistically significant difference from IL-1β values. #P<0.001 (Student t test). Osteoarthritis and Cartilage 2005 13, 243-249DOI: (10.1016/j.joca.2004.11.008) Copyright © 2004 OsteoArthritis Research Society International Terms and Conditions