Anti-β-actin antibodies, 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), dexamethasone (DEX), formaldehyde, insulin, HEPES, Nycodenz, propidium iodide (PI), and Triton X-100 were purchased from Sigma-Aldrich (Missouri, USA). Fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific Inc. (NY, USA). Percoll and pronase were purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Trypan blue solution (0.5%) was purchased from BioWest (Nuaille, France). All other reagents were purchased from Invitrogen (CA, USA).
Pronase
Manufactured by GE Healthcare
Sourced in Sweden
Pronase is a proteolytic enzyme derived from the extracellular secretions of the bacterium Streptomyces griseus. It functions as a non-specific protease, capable of hydrolyzing a wide range of protein substrates.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
Nycodenz, by Merck Group (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions)
Trypan blue solution, by Biowest (1 mentions)
Vivaspin column, by GE Healthcare (1 mentions)
2 protocols using pronase
1
Isolation and Culture of Primary Cells
2
Glycosaminoglycan Extraction from Conditioned Medium
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Cells underwent starvation in growth medium without serum for 24 h at 37°C with 5% CO2 in air. Then, 10 mL of conditioned medium (CM) were centrifuged to remove cell debris. CM supernatants were concentrated using a Vivaspin™ column with a 10 000 Da MWCO (GE Healthcare Bio‐Sciences AB), and incubated overnight at 37°C with a pronase (Cat. No. P8811‐1G, Sigma‐Aldrich) to digest all proteins. Proteinase deactivation was performed by addition of NaCl (50 nmol/L) and incubation at 100°C for 1 min. After cooling, centrifugation was performed to pellet digested proteins. GAGs were precipitated from the supernatant by addition of saturated sodium acetate and incubation at 4°C for 3 h. Precipitated GAGs were air‐dried and resuspended in sterile distilled water. For each cell line, 3 independent biological replicates were analyzed.
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