The zebrafish heart including atrium, ventricle and bulbus arteriosus was transferred into cold HBSS and opened carefully with forceps, allowing most of the erythrocytes to be washed away. Afterwards, the heart tissue was transferred into 500 µl HBSS containing Liberase™ enzyme mix (Sigma-Aldrich, 0.26 U/mL final concentration) and Pluronic® F-68 (Thermo Fisher Scientific, 0.1 %). The reaction was incubated at 37°C for 30 minutes while shaking at 750 rpm with intermittent pipette mixing. Afterwards, most of the tissue was dissociated. The reaction was stopped by adding 500 µl cold HBSS supplemented with 1% BSA. The cells were pelleted by centrifuging at 200 g in a table-top centrifuge at 4°C, then washed and filtered following the procedure described above for 5 dpf larvae.
Liberase enzyme mix
Manufactured by Merck Group
Liberase™ enzyme mix is a proprietary blend of highly purified enzymes developed by Merck Group for the isolation and dissociation of cells from a variety of tissue types. The product facilitates the gentle and efficient release of cells from the extracellular matrix, enabling the extraction of high-quality single-cell suspensions for downstream applications.
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Lab products found in correlation
3 protocols using liberase enzyme mix
1
Dissociation of Zebrafish Cardiac Tissue
2
Dissociation of Zebrafish Cardiac Tissue
3
Isolating Zebrafish Heart Single Cells
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Adult zebrafish were humanely killed at different times post-injury by placing them in ice cold water of 0-4°C for 20 minutes. The heart was dissected from the fish and transferred into cold HBSS. A needle and a syringe filled with cold HBSS were used to pierce into the lumen of the heart to thoroughly wash away most of the erythrocytes in the tissue. Afterwards, the tissue was opened carefully with forceps, and the heart tissue was incubated at 37°C for 30 min in 500µl HBSS containing Liberase enzyme mix (Sigma-Aldrich, 0.26 U/mL final concentration) and Pluronic F-68 (Thermo Fisher Scientific, 0.1%), while shaking at 750 r.p.m. with intermittent pipette mixing. After most of the tissue was dissociated, the reaction was stopped by adding 500µl cold HBSS supplemented with 1% BSA. The suspension was centrifuged at 250g at 4°C and washed two times with 500µl cold HBSS containing 0.05% BSA, then filtered through a cell strainer of 35µm diameter.
The quality of the single cell suspension was then confirmed under the microscope, and cells were counted prior to scRNA-seq library preparation.
The quality of the single cell suspension was then confirmed under the microscope, and cells were counted prior to scRNA-seq library preparation.
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