The animals were sacrificed immediately after cutting the spinal cord (cervical dislocation). The tissue separated from the animal was placed in the sterile Petri dish, the lipid and lymph glands were separated by stereomicroscope (CETI), and part of the tissue was removed for aldehyde fuchsin staining. The other sections of the tissue were cut into 2 mm segments by a surgical blade and 10 mL collagenase type H (Roche) with concentration of 4 mg/mL was added to it and kept in incubator for 20 min. The undigested segments were separated by Pasture pipette and washed several times with HBSS containing 20% fetal calf serum (Gibco) and centrifuged for 1 min in 200 g each time. The obtained solution was cultured for 24 h in RPMI-1640 medium (Sigma) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1% penicillin–streptomycin (Sigma), and incubated at 37°C and in humidified atmosphere containing 5% CO2.[15 (link)]
Collagenase type h
Manufactured by Roche
Sourced in Germany
Collagenase type H is an enzyme that is used to break down collagen, a major structural component of the extracellular matrix. It is commonly used in cell and tissue isolation and dissociation procedures.
Automatically generated - may contain errors
Lab products found in correlation
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Trypsin like enzyme, by Thermo Fisher Scientific (1 mentions)
Countess hemocytometer, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
70 μm cell strainer, by BD (1 mentions)
2 protocols using collagenase type h
1
Isolation and Culture of Tissue Cells
2
Isolation and Expansion of Subcutaneous Adipose-Derived Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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A subcutaneous fat biopsy (approximately 2–4 g) was obtained under sterile conditions from RVD patients in an outpatient surgical suite or during the surgical nephrectomy from kidney donors. Cells were expanded ex vivo as follows. The fat tissues were minced with surgical scalpels and incubated in 2 % collagenase type H (Roche, Mannheim, Germany) for 90 min at 37 °C. Digested tissue was centrifuged at 400 g for 5 min with the pellet washed in PBS, passed through a 70-μm cell strainer (BD Biosciences, San Jose, CA, USA), and incubated in red blood cell lysis buffer (154 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA). Cells were grown in T-75 cm2 flasks at a concentration of 1.0–2.5 × 103 cells/cm2 in Advanced MEM with 5 % PLTmax (Mill Creek Life Sciences, Rochester, MN, USA) and 2 mM L-glutamine (Invitrogen, Carlsbad, CA, USA) in a 37 °C 5 % CO2 incubator for 3–4 days. When cells were 60–80 % confluent, they were passaged using TrypLE (Trypsin-like Enzyme, Invitrogen) [15 (link)]. Cell yield was quantified with a Countess hemocytometer (Thermo Fisher Scientific, Inc., NY, USA).
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