Isolation of Colon Mononuclear Cells

Mononuclear cells were isolated as described previously with minor modifications^{9 (link)}. The colon was flushed, cut open longitudinally and incubated in 5 mM EDTA and 10% FBS-containing HBSS buffer to wash out intraepithelial mononuclear cells. The colon was then cut into small pieces, incubated in 5 ml digesting buffer contained 500 U collagenase from Clostridium histolyticum (C2139, Sigma-Aldrich), 2.5 μg DNase I (10104159001, Roche) and 10% FBS (Gibco) at 37 °C for 2 h. The digested colon was homogenized using a 70 μm strainer. CD45⁺ cells were isolated using a CD45 magnetic beads isolation kit (130-052-301, Miltenyi Biotec).

Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link: Access Free Full Text.

Sanmarco L.M., Chao C.C., Wang Y.C., Kenison J.E., Li Z., Rone J.M., Rejano-Gordillo C.M., Polonio C.M., Gutierrez-Vazquez C., Piester G., Plasencia A., Li L., Giovannoni F., Lee H.G., Akl C.F., Wheeler M.A., Mascanfroni I., Jaronen M., Alsuwailm M., Hewson P., Yeste A., Andersen B.M., Franks D.G., Huang C.J., Ekwudo M., Tjon E.C., Rothhammer V., Takenaka M., de Lima K.A., Linnerbauer M., Guo L., Covacu R., Queva H., Fonseca-Castro P.H., Bladi M.A., Cox L.M., Hodgetts K.J., Hahn M.E., Mildner A., Korzenik J., Hauser R., Snapper S.B, & Quintana F.J. (2022). Identification of environmental factors that promote intestinal inflammation. Nature, 611(7937), 801-809.

Publication 2022

collagenase from Clostridium histolyticum DNase I FBS CD45 magnetic beads isolation kit

Buffer Cells Clostridium histolyticum collagenase Colon Dnase i Edta Isolation

Corresponding Organization :

Other organizations : Brigham and Women's Hospital, Harvard University, National Yang Ming Chiao Tung University, Woods Hole Oceanographic Institution, Max Delbrück Center, Boston Children's Hospital, Broad Institute

Top 5 similar protocols

Variable analysis

independent variables

Extraction method for mononuclear cells from the colon
Incubation time of colon tissue in digesting buffer

dependent variables

Isolation of CD45+ cells

control variables

Concentration of EDTA in buffer
Concentration of FBS in buffer
Concentration of collagenase in digesting buffer
Concentration of DNase I in digesting buffer

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!