Isolation and Culture of Cancer-Associated Fibroblasts
CAFs were isolated from MMTV+;FSP-Cre−;FAKfl/fl and MMTV+;FSP-Cre+;FAKfl/fl mice as described previously62 (link). Tumours were cut into small pieces and digested for 1 h at 37 °C in 5 mL collagenase/dispase (1 mg/mL; Sigma). After filtering the undigested tissue, the solution was centrifuged (3000 g, 5 min) and the final pellet was resuspended in DMEM high glucose, 10% FBS, 1% ITS and seeded on a 6 cm dish. After 30 min, fibroblasts are adhered and non-adherent tumour cells were replated and cultured as described above. Adherent fibroblasts were used after 1–3 passages.
Other organizations :
Queen Mary University of London, MRC Cancer Unit, Hutchison/MRC Research Centre, University of Cambridge, ChemoCentryx (United States), Hospital Universitario Miguel Servet, Instituto de Investigación Sanitaria Aragón
Genotype (MMTV+;FSP-Cre−;FAKfl/fl vs. MMTV+;FSP-Cre+;FAKfl/fl)
dependent variables
Characteristics of isolated cancer-associated fibroblasts (CAFs)
control variables
Collagenase/dispase concentration (1 mg/mL)
Incubation time for tissue digestion (1 h)
Incubation temperature for tissue digestion (37 °C)
Cell culture media (DMEM high glucose, 10% FBS, 1% ITS)
Passage number of adherent fibroblasts (1-3 passages)
controls
Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.
Annotations
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