Retinas were dissected in fresh cold (4 °C) 1X Dulbecco’s PBS (DPBS; D8537–500ML, Sigma-Aldrich), transferred to a 5 ml polypropylene round-bottom tube, and promptly dissociated with a commonly used papain dissociation protocol (Worthington Biochemicals) with minor modifications. Briefly, single retinas were incubated in 1 ml of digestion solution at 37 °C for 10 min. Mechanical trituration was performed by pipetting 30 times with a P1000 pipette. Cells were centrifuged, using a swing-bucket rotor, at 300 ×g for 5 min at 4 °C, and the digestion solution was removed. The dissociated cells were gently resuspended in 700 µl of inactivation solution prewarmed for 10 min at 28 °C. Then, 700 µl of chilled washing solution was layered under the cell suspension, and the cells were centrifuged at 300 ×g for 5 min at 4 °C. After the washing solution was removed, cells were resuspended in 500 µl of DPBS containing 0.04% BSA and passed through a 40 μm cell strainer (pluriSelect, Leipzig, Germany).
Papain dissociation protocol
Manufactured by Worthington
The Papain dissociation protocol is a lab equipment product designed for the gentle dissociation of cells and tissues. It utilizes the enzyme papain to break down the extracellular matrix, allowing for the isolation of individual cells or cell clusters. This protocol is commonly used in cell culture, tissue engineering, and other applications requiring the separation of cells from their surrounding environment.
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2 protocols using papain dissociation protocol
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Dissociation of Retinal Cells
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Papain Dissociation for Single-cell RNA-seq
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We collected three organoids per samples and dissociated using the papain dissociation protocol (Worthington Biochemicals) according to the manufacturer’s protocol. Briefly, organoids were incubated for 30 min at 37 °C in Earle’s Balanced Salt Solution (EBSS)/Albumin-ovomucoid inhibitor/papain with gentle shaking and mechanical dissociation. The pellet was resuspended and incubated with (EBSS)/Albumin-ovomucoid inhibitor/DNAse for 2 min followed by incubation with ovomucoid for 2 min. The cells were washed and resuspended with DMEM/F12. After determination of the cell density and viability, cells were submitted to single-cell RNA sequencing (10X Genomics, Chromium Single cell 3’ v3) to recover ~5000 sequenced cells per sample with more than 50,000 reads per cell. We have uploaded the RNAseq data used in this paper in NCBI’s Gene Expression Omnibus under accession code GSE174569.
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