DCs and TECs were harvested from thymus by treatment with 2 mL of collagenase IV (0.1% m/v in PBS) and DNaseI (20 μg/mL in PBS; Life Technologies) for 10 minutes at 37 degrees C at 150 rpm, followed by a second round of collagenase IV/DNaseI digestion. Next, the homogenate was further digested twice with 2 mL collagenase dispase (0.12% m/v in PBS; Life Technologies) and 20 μg/mL DNaseI, at 37°C, 150 rpm. Thymic cells were obtained following two 10 minute incubations with 2 mL of 0.1% collagenase IV and two 10 minute incubations with 2 mL of 0.12% collagenase dispase. Remaining tissue was pushed through 40 μm pore nylon mesh (BD Biosciences) to obtain a single cell suspension of thymocytes, including TECs and DCs. Total cell counts were obtained using a hemocytometer.
Collagenase dispase
Manufactured by Thermo Fisher Scientific
Collagenase dispase is a enzymatic reagent used for the dissociation and disaggregation of tissues and cells. It contains a combination of collagenase and dispase enzymes that break down the extracellular matrix, facilitating the isolation of individual cells from tissues.
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Lab products found in correlation
2 protocols using collagenase dispase
1
Isolation of Thymic DCs and TECs
2
Thymic Cell Isolation Protocol
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DCs and TECs were collected from thymus by treatment with 2 ml of collagenase IV (0.1% m/v in phosphate-buffered saline (PBS)) and DNaseI (20 μg ml−1 in PBS; Life Technologies, Burlington, ON, Canada) for 10 min at 37 °C at 150 r.p.m., followed by a second round of collagenase IV/DNaseI digestion. Next, the homogenate was further digested twice with 2 ml collagenase dispase (0.12% m/v in PBS; Life Technologies) and 20 μg ml−1 DNaseI, at 37 °C, 150 r.p.m. Thymic cells were obtained following two 10 min incubations with 2 ml of 0.1% collagenase IV and two 10 min incubations with 2 ml of 0.12% collagenase dispase. Remaining tissue was pushed through 40 μm pore nylon mesh (BD Biosciences, Mississauga, ON, Canada) to obtain a single-cell suspension of thymocytes, including TECs and DCs. Total cell counts were obtained using a hemocytometer.
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