Collagenase 4 ls004188

For hematoxylin and eosin (H&E) staining, entire lung tissues (4T1 mouse model) were collected from each mouse at sacrifice and formalin-fixed and paraffin-embedded (FFPE). For flow cytometry analysis or detection of lung metastatic cancer cells using cell culture techniques, each tumor or lung (50% tissue of each lung, MCa-M3C mouse model), and spleen was collected at sacrifice. Primary tumors or lungs were minced into 3 × 3 mm pieces and digested with Collagenase IV (LS004188) (1 mg/mL PBS) (Worthington Biochemical Corp, Lakewood, NJ, USA.) at 37 °C for 1 h. Spleens were mechanically homogenized using a fine metal mesh net and then the red blood cells in the splenocyte suspension were lysed with ammonium-chloride-potassium (ACK) lysing. The digested tumor or splenocyte suspension was filtered through a 40 μM cell strainer to obtain single-cell suspensions for flow analysis.

For hematoxylin and eosin (H&E) staining, entire lung tissues (4T1 muse model) were collected from each mouse at sacrifice and formalin-fixed and paraffin-embedded (FFPE). For flow cytometry analysis or detection of lung metastatic cancer cells using cell culture techniques, each tumor or lung (50% tissue of each lung, MCa-M3C mouse model), and spleen was collected at sacrifice. Primary tumors or lungs were minced into 3 × 3 mm pieces and digested with Collagenase IV (LS004188) (1 mg/mL PBS) (Worthington Biochemical Corp.) at 37°C for 1 h. Spleens were mechanically homogenized using a fine metal mesh net and then the red blood cells in the splenocyte suspension were lysed with ammonium-chloride-potassium (ACK) lysing. The digested tumor or splenocyte suspension were filtered through a 40 μM cell strainer to obtain single-cell suspensions for flow analysis.

Hank's Balanced Salt Solution (HBSS, 14025092), Opti-Minimal Essential Medium (Opti-MEM; 31985070), and Roswell Park Memorial Institute (RPMI) 1640 medium (11875093) were purchased from Thermo Fisher Scientific (Waltham, MA). Bovine serum albumin (BSA; A9418) was obtained from Sigma-Aldrich (St. Louis, MO), and collagenase IV (LS004188) was sourced from Worthington Biochemical Corporation (Lakewood, NJ). U0126 (S1102), PD98059 (S1177), SP600125 (S1460), and SB203580 (S1076) were purchased from Selleckchem (Houston, TX). Recombinant human TNFα (300-01A) was obtained from PeproTech (Rocky Hill, NJ). Ficoll-Paque PLUS (17-5442-02) was purchased from GE Healthcare (Marlborough, MA). Mouse anti-human MMP3 antibody (Ab; 1B4, sc-21732) and mouse anti-human β-actin (C4, sc-47778) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX). Rabbit antibodies for the following proteins and phosphoproteins were purchased from Cell Signaling Technology (Beverly, MA): phospho-p44/42 mitogen-activated protein kinase (MAPK; T202/Y204) (9101), p44/42 MAPK (Erk1/2) (9102), phospho-SAPK/c-Jun N-terminal kinase (JNK) (T183/Y185) (9251), SAPK/JNK (9252), phospho-p38 MAPK (T180/Y182) (9211), and p38 MAPK (9212).