Colonic Crypt Isolation Protocol

Colonic crypt isolation was performed.⁴⁸ (link) Briefly, isolated colonic tissues were chelated in buffer consisting of 3 mM EDTA (Corning) and 0.5mM DTT (Teknova) in 1X Dulbecco’s Buffered Saline (DPBS) for 1 hour and 15 minutes rotating at 4°C. The tissue was then transferred to 1X DPBS and shaken rigorously for 2 minutes to separate the colonic epithelium from the tissue. After transfer of crypts to a new tube, shaking was repeated 3X to collect remaining crypts. Crypts were washed in DPBS and divided among various enzymic cocktails and conditions for dissociation.

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Arceneaux D., Chen Z., Simmons A.J., Heiser C.N., Southard-Smith A.N., Brenan M.J., Yang Y., Chen B., Xu Y., Choi E., Campbell J.D., Liu Q, & Lau K.S. (2023). A contamination focused approach for optimizing the single-cell RNA-seq experiment. iScience, 26(7), 107242.

Publication 2023

EDTA DTT

Colonic Crypts Dissociation conditions Edta Enzymic Epithelium Isolation Saline Tissue

Corresponding Organization : Vanderbilt University

Other organizations : James S. McDonnell Foundation, Washington University in St. Louis, 1Cellbio (United States), Boston University

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Variable analysis

independent variables

Buffer consisting of 3 mM EDTA (Corning) and 0.5mM DTT (Teknova) in 1X Dulbecco's Buffered Saline (DPBS)

dependent variables

Separation of colonic epithelium from the tissue
Collection of colonic crypts

control variables

Temperature at 4°C
Duration of chelation for 1 hour and 15 minutes
Duration of shaking for 2 minutes and repeated 3 times

controls

No positive or negative controls were explicitly mentioned in the provided information.

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